KR102490952B1 - Intracellular delivery of biomolecules that induce tolerance - Google Patents

Abstract

The present invention is a method for inducing tolerance to an antigen or suppressing an immune response by passing a cell suspension containing immune cells through a constriction, wherein the constriction transforms the cells, causing disturbance of the cells, thereby reducing antigen and/or tolerogenicity. wherein the factor enters the cell, thereby generating tolerogenic and/or immunosuppressive immune cells. In some embodiments, tolerogenic and/or immunosuppressive immune cells are delivered to an individual and presentation of the antigen induces tolerance to the antigen and/or suppresses an immune response.

Description

Intracellular delivery of biomolecules that induce tolerance

Cross-Reference to Related Applications

This application claims priority to U.S. Provisional Application No. 62/331,384, filed on May 3, 2016, which application is hereby incorporated by reference in its entirety.

field of invention

Generally, the present disclosure relates to a method of suppressing an immune response or inducing tolerance by delivering a compound into a cell by passing a cell suspension through a cell-modifying constriction.

Undesirable immune responses contribute to autoimmunity, transplant rejection, allergy, and antidrug responses. Autoimmunity develops when an organism initiates an anti-autologous response, usually as a result of a dysregulated immune response to self-antigens. Autoimmune diseases include, for example, type I diabetes, systemic lupus erythematosus, rheumatoid arthritis, autoimmune hemolytic anemia, and multiple sclerosis. A pathogenic immune response in the recipient organism following transplantation of a donor organ can lead to transplant rejection and reduced patient survival. Additionally, unwanted immune responses to food and environmental antigens lead to allergic diseases such as asthma, food allergies, and atopic dermatitis. Therefore, approaches to establish immunological tolerance to an antigen are the focus of intensive therapy development.

Current intracellular delivery methods are not effective in modulating cell phenotype or function to induce antigen-specific tolerance. Therefore, there is a need for intracellular delivery technologies capable of loading antigens and tolerogenic factors into the cytoplasm of cells and driving potent immunosuppressive responses for the treatment of pathogenic immune responses underlying autoimmune diseases and transplant rejection. not met References describing methods of delivering compounds to cells using microfluidic constriction include WO2013059343, WO2015023982, WO2016070136, WO2016077761, and PCT/US2016/13113.

All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

The present invention is a method for inducing challenge to an antigen in a subject, wherein a cell suspension containing immune cells is passed through a constriction, wherein the constriction transforms the cells to cause cell disruption so that the antigen and/or tolerogenic factor become immune. entering a cell, wherein tolerogenic immune cells are produced, and introducing the immune cells into a subject. In some embodiments, tolerogenic immune cells are capable of inducing tolerance to an antigen, eg, by presentation of said antigen by said tolerogenic immune cells. In some embodiments, the tolerogenic factor modulates the activity and/or expression of a costimulatory molecule, modulates the activity and/or expression of an immunosuppressive molecule, and/or modulates the activity and/or expression of an inflammatory molecule. and/or modulates the activity of another immune cell.

A particular aspect of the invention is a method of suppressing an immune response in a subject, wherein a cell suspension comprising immune cells is passed through a constriction, wherein the constriction transforms the cells, thereby causing disruption of the cells so that antigens and/or tolerogenic factors are released. Entering an immune cell, wherein an immunosuppressive immune cell is generated, and introducing the immune cell into a subject. In some embodiments, presentation of said antigen by said immunosuppressive immune cells suppresses an immune response to the antigen.

A particular aspect of the invention is a method of inducing tolerance to an antigen in a subject comprising introducing immune cells into the subject, wherein the immune cells comprise an antigen and/or tolerogenic factor, wherein the antigen and tolerogenic factor is introduced into the immune cells by passing the immune cells through the constriction, wherein the constriction transforms the cells to cause disruption of the cells so that antigens and/or tolerogenic factors enter the immune cells.

A particular aspect of the invention is a method of suppressing an immune response in a subject comprising introducing immune cells into the subject, wherein the immune cells comprise an antigen and/or tolerogenic factor, wherein the antigen and tolerogenic factor comprise an immune cell. Introduction of the immune cells by passing the cells through a constriction, wherein the constriction transforms the cells, causing disruption of the cells so that antigens and/or tolerogenic factors enter the immune cells.

A particular aspect of the invention is a method of generating tolerogenic immune cells, comprising passing a cell suspension comprising immune cells through a constriction, wherein the constriction modifies the cells, thereby causing disturbance of the cells so that the tolerogenic factor is immune. and enters cells, thereby producing tolerogenic immune cells.

A particular aspect of the invention is a method of generating immunosuppressive immune cells, comprising passing a cell suspension comprising immune cells through a constriction, wherein the constriction modifies the cells, thereby causing disturbance of the cells, so that tolerogenic factors are and enters immune cells, thereby generating immunosuppressive immune cells.

A particular aspect of the invention is a method of generating tolerogenic antigen-presenting immune cells, wherein the tolerogenic immune cells further pass through a second constriction, wherein the second constriction transforms the cells, thereby causing perturbation of the cells to antigen It provides a method of entering the immune cell. In some embodiments, antigens are presented by tolerogenic immune cells.

A particular aspect of the invention is a method of generating immunosuppressive antigen-presenting immune cells, wherein the immunosuppressive immune cells further pass through a second constriction, wherein the second constriction modifies the cells and thereby disrupts the cells. causing antigens to enter immune cells. In some embodiments, antigens are presented by immunosuppressive immune cells.

A particular aspect of the invention is a method of generating tolerogenic antigen-presenting cells, wherein the antigen-presenting cells pass through a constriction, wherein the constriction causes perturbation of the cells by modifying the cells so that tolerogenic factors are introduced into the antigen-presenting cells. It provides a way to enter. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule). In some embodiments, antigen-presenting cells are generated in vitro or in vivo prior to introduction of the immunosuppressive molecule.

A particular aspect of the invention is a method of generating immunosuppressive antigen-presenting cells, wherein the antigen-presenting cells pass through a constriction, wherein the constriction transforms the cells, causing perturbation of the cells so that the tolerogenic factor is antigen-presenting. A method of entering a cell is provided. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule). In some embodiments, antigen presenting cells are generated in vitro or in vivo prior to introduction of tolerogenic factors.

A particular aspect of the invention is a method of delivering tolerogenic factors that produce an tolerogenic phenotype into immune cells, comprising passing a cell suspension comprising immune cells through a constriction, wherein the constriction modifies the immune cells so that the cells causing perturbation of the tolerogenic factor to enter the cell, wherein the cell suspension is contacted with the tolerogenic factor. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule).

A particular aspect of the invention is a method of delivering tolerogenic factors that produce an immunosuppressive phenotype into immune cells, comprising passing a cell suspension comprising immune cells through a constriction, wherein the constriction is caused by modifying the immune cells. Tolerogenic factors that cause perturbation of the cells to produce an immunosuppressive phenotype enter the cells, wherein the cell suspension is contacted with the tolerogenic factors. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule).

A particular aspect of the invention is a method of suppressing an immune response in a subject, wherein a first cell suspension comprising a first immune cell is passed through a constriction, wherein the constriction modifies the cells, thereby causing disturbance of the cells so that an antigen is released from the immune cells. wherein the second cell suspension comprising second immune cells is passed through the constriction, wherein the constriction transforms the cells to cause disturbance of the cells so that the tolerogenic factor enters the immune cells, wherein immunosuppression wherein sexual immune cells are produced, and introducing the first and second immune cells into a subject. In some embodiments, presentation of said antigen by said first or second immune cell suppresses an immune response to the antigen. In some embodiments, the first immune cells and the second immune cells are introduced into the subject simultaneously. In some embodiments, a first immune cell and a second immune cell are introduced sequentially into an individual. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule).

A particular aspect of the invention is a method of inducing tolerance to an antigen in a subject by passing a first cell suspension comprising a first immune cell through a constriction, wherein the constriction transforms the cells thereby causing disruption of the cells so that the antigen becomes entering immune cells, passing a second cell suspension comprising second immune cells through the constriction, wherein the constriction causes disturbance of the cells by modifying the cells so that tolerogenic factors enter the immune cells, wherein Tolerogenic immune cells are produced, and introducing the first and second immune cells into a subject. In some embodiments, presentation of said antigen by said first or second immune cell induces tolerance to the antigen. In some embodiments, the first immune cells and the second immune cells are introduced into the subject simultaneously. In some embodiments, a first immune cell and a second immune cell are introduced sequentially into an individual. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule).

A particular aspect of the invention is a method of suppressing an immune response in a subject, wherein a cell suspension comprising immune cells is passed through a constriction, wherein the constriction deforms the cells, causing perturbation of the cells, thereby encoding a non-functional cytokine binding protein. wherein a compound that causes entry into an immune cell, and introducing the immune cell into a subject, wherein the non-functional cytokine binding protein is expressed, wherein the non-functional cytokine binding protein binds to a free inflammatory cytokine. binding and thereby suppressing the immune response, for example by inhibiting inflammatory immune cells. In some embodiments, the non-functional cytokine binding protein comprises a non-functional cytokine receptor. In some embodiments, the non-functional cytokine receptor lacks a cytoplasmic signaling domain. In some embodiments, the non-functional cytokine binding protein comprises a proteolytic site that cleaves a target cytokine. In some embodiments, the non-functional cytokine binding protein comprises an anti-cytokine antibody. In some embodiments, the non-functional cytokine binding protein comprises an anti-cytokine B cell receptor.

In some embodiments that may be combined with the previous embodiments, the immune cells are from an individual. In some embodiments, the immune cells are from different individuals. In some embodiments, the immune cells are from a pool of cells from multiple individuals.

In some embodiments that may be combined with the previous embodiments, the constriction is contained within the microfluidic channel. In some embodiments, the constriction is a pore or is contained within a pore. In some embodiments, the pores are contained within the surface. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane. In some embodiments, constriction size is a function of immune cell diameter. In some embodiments, the constriction size is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter. In some embodiments, the channel comprises a constriction length of about 10 μm and a constriction width of about 4 μm. In some, the pore size is about 0.4 μm, about 3 μm, about 4 μm, about 5 μm, about 8 μm, about 10 μm, about 12 μm, or about 14 μm. In some embodiments, the method is performed at about -5°C to about 45°C.

Unless otherwise specified, reference to the diameter of an unnucleated cell refers to the diameter of a cell in fluid prior to passing through the constriction, eg, as the cell approaches the constriction.

In some embodiments that may be combined with the previous embodiments, the cell suspension comprises a mixed cell population. In some embodiments, the cell suspension is whole blood. In some embodiments, the cell suspension comprises peripheral blood mononuclear cells. In some embodiments, a cell suspension comprises a purified cell population. In some embodiments, a cell suspension comprises mammalian cells. In some embodiments, the cell suspension comprises monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cells. In some embodiments, a cell suspension comprises human cells. In some embodiments, a cell suspension comprises non-mammalian cells. In some embodiments, the cell suspension comprises bacterial, yeast, chicken, frog, insect, fish, or nematode cells. In some embodiments, the immune cells are mammalian cells. In some embodiments, the immune cells are monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cells. In some embodiments, the immune cells are human cells. In some embodiments, the immune cell is a T cell, B cell, dendritic cell, monocyte, macrophage, NK cell, innate lymphoid cell, neutrophil, basophil, eosinophil, bone marrow derived suppressor cell, or mast cell. In some embodiments, an antigen-presenting cell is a mammalian cell. In some embodiments, the antigen-presenting cells are monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cells. In some embodiments, an antigen-presenting cell is a human cell. In some embodiments, the antigen-presenting cell is a T cell, B cell, dendritic cell, monocyte, macrophage, NK cell, innate lymphoid cell, neutrophil, basophil, eosinophil, bone marrow derived suppressor cell, or mast cell.

In some embodiments that may be combined with the previous embodiments, the antigen is a foreign antigen. In some embodiments, the antigen is a self-antigen. In some embodiments, the antigen is an allograft transplant antigen. In some embodiments, the antigen is a modified antigen. In some embodiments, a modified antigen comprises an antigen fused with a therapeutic agent. In some embodiments, a modified antigen comprises an antigen fused with a targeting peptide. In some embodiments, the cell suspension is contacted with the antigen before, concurrently with, or after passage through the stenosis.

In some embodiments that may be combined with the previous embodiments, the tolerogenic factor inhibits the activity of the costimulatory molecule. In some embodiments, the tolerogenic factor reduces expression of costimulatory molecules. In some embodiments, the tolerogenic factor deletes a nucleic acid that modulates expression of a costimulatory molecule. In some embodiments, the tolerogenic factor inhibits a costimulatory molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that inhibits the expression of costimulatory molecules. In some embodiments, the tolerogenic factor increases the activity of a protein inhibitor that inhibits expression of costimulatory molecules. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an inhibitor of a costimulatory molecule. In some embodiments, the costimulatory molecule is CD80 or CD86.

In some embodiments that can be combined with the previous embodiments, the tolerogenic factor potentiates the activity of an immunosuppressive factor. In some embodiments, the immunosuppressive factor is a co-inhibitory molecule, transcriptional regulator, or immunosuppressive molecule. In some embodiments, the tolerogenic factor potentiates the activity of a co-inhibitory molecule. In some embodiments, the tolerogenic factor increases expression of co-inhibitory molecules. In some embodiments, the tolerogenic factor encodes a co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the activity of the co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that enhances the expression of co-inhibitory molecules. In some embodiments, the tolerogenic factor increases the activity of the polypeptide to increase expression of co-inhibitory molecules. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an enhancer of a co-inhibitory molecule. In some embodiments, the co-inhibitory molecule is PD-L1, PD-L2, or CTLA-4.

In some embodiments, the tolerogenic factor enhances the activity of an immunosuppressive molecule. In some embodiments, tolerogenic factors increase expression of immunosuppressive molecules. In some embodiments, the tolerogenic factor encodes an immunosuppressive molecule. In some embodiments, the tolerogenic factor increases the activity of an immunosuppressive molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that enhances the expression of immunosuppressive molecules. In some embodiments, the tolerogenic factor increases the activity of the polypeptide to enhance expression of immunosuppressive molecules. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an enhancer of an immunosuppressive molecule. In some embodiments, the immunosuppressive molecule is arginase-1 (ARG1), nitric oxide (NO), nitric oxide synthase 2 (NOS2), indoleamine 2,3-dioxygenase (IDO), IL-4 , IL-10, IL-13, IL-35, IFNα, or TGFβ.

In some embodiments, the tolerogenic factor inhibits the activity of an inflammatory molecule. In some embodiments, the inflammatory molecule is an inflammatory transcription factor. In some embodiments, the tolerogenic factor inhibits an inflammatory transcription factor. In some embodiments, the tolerogenic factor reduces expression of an inflammatory transcription factor. In some embodiments, the tolerogenic factor deletes a nucleic acid encoding an inflammatory transcription factor. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that inhibits the expression of an inflammatory transcription factor. In some embodiments, the tolerogenic factor increases the activity of a protein inhibitor that inhibits the expression of an inflammatory transcription factor. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an inhibitor of an inflammatory transcription factor. In some embodiments, the inflammatory transcription factor is a molecule associated with NF-κB, an interferon regulator, or the JAK-STAT signaling pathway. In some embodiments, the tolerogenic factor encodes a modified TCR containing a cytoplasmic signaling domain that upon binding to antigen triggers the production of immunosuppressive cytokines. In some embodiments, the tolerogenic factor encodes a chimeric antigen receptor that contains a cytoplasmic signaling domain that upon binding to the antigen triggers the production of immunosuppressive cytokines.

In some embodiments that can be combined with the previous embodiments, the tolerogenic factor comprises a nucleic acid. In some embodiments, tolerogenic factors include nucleic acids encoding siRNA, mRNA, miRNA, lncRNA, tRNA, or shRNA. In some embodiments, the tolerogenic factor is a plasmid. In some embodiments, the tolerogenic factor comprises a protein-nucleic acid complex. In some embodiments, the tolerogenic factor comprises a Cas polypeptide and a guide RNA or donor DNA. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding a Cas polypeptide and a guide RNA or donor DNA. In some embodiments, the tolerogenic factor comprises a Cas9 polypeptide and a guide RNA or donor DNA. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding a Cas9 polypeptide and a guide RNA or donor DNA. In some embodiments, the tolerogenic factor comprises a polypeptide. In some embodiments, the polypeptide is a nuclease, TALEN protein, zinc finger nuclease, meganuclease, or CRE recombinase. In some embodiments, the polypeptide is a transposase or integrase enzyme. In some embodiments, a polypeptide is an antibody. In some embodiments, a polypeptide is a transcription factor. In some embodiments, the tolerogenic factor is a small molecule. In some embodiments, the tolerogenic factor is a nanoparticle. In some embodiments, the cell suspension is contacted with the tolerogenic factor before, concurrently with, or after passage through the stenosis.

In embodiments in which the immune response is suppressed, the immune response is suppressed by at least about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, or greater than about 200%. In some embodiments, suppressing an immune response comprises reducing a T cell response. In some embodiments, reducing a T cell response comprises reducing T cell activation. In some embodiments, reducing a T cell response comprises reducing T cell survival. In some embodiments, reducing a T cell response comprises reducing T cell proliferation. In some embodiments, reducing a T cell response comprises reducing T cell functionality. In some embodiments, a decrease in T cell response comprises a change in T cell phenotype. In some embodiments, suppressing an immune response comprises non-co-stimulatory activation of T cells. In some embodiments, suppressing an immune response comprises enhancing a Treg response. In some embodiments, suppressing an immune response comprises reducing a B cell response. In some embodiments, reducing a B cell response comprises reducing antibody production. In some embodiments, suppressing an immune response comprises reducing cytokine production. In some embodiments, suppressing an immune response comprises reducing an autoimmune response. In some embodiments, suppressing an immune response comprises reducing an allergic response. In some embodiments, the antigen is an antigen associated with the transplanted tissue. In some embodiments, suppressing an immune response comprises reducing an immune response to the transplanted tissue. In some embodiments, the antigen is associated with a virus. In some embodiments, suppressing an immune response comprises reducing a pathogenic immune response to a virus. In some embodiments, an antigen is associated with a bacterium. In some embodiments, suppressing an immune response comprises reducing a pathogenic immune response to a bacterium. In some embodiments, the antigen is associated with a fungus. In some embodiments, suppressing the immune response comprises reducing a pathogenic immune response to the fungus. In some embodiments, suppressing an immune response comprises reducing an immune response to a therapeutic agent. In some embodiments, suppressing the immune response comprises reducing the immune response to the therapeutic vehicle.

In embodiments in which tolerance is induced, tolerance may include a decrease in T cell response. In some embodiments, reducing a T cell response comprises reducing T cell activation. In some embodiments, reducing a T cell response comprises reducing T cell survival. In some embodiments, reducing a T cell response comprises reducing T cell proliferation. In some embodiments, reducing a T cell response comprises reducing T cell functionality. In some embodiments, a decrease in T cell response comprises a change in T cell phenotype. In some embodiments, tolerance includes non-costimulatory activation of T cells. In some embodiments, tolerance comprises enhancing Treg responses. In some embodiments, tolerance comprises reducing a B cell response. In some embodiments, reducing a B cell response comprises reducing antibody production. In some embodiments, tolerance comprises a decrease in cytokine production. In some embodiments, tolerance includes reducing an autoimmune response. In some embodiments, tolerance includes reducing an allergic response. In some embodiments, tolerance comprises a reduced immune response to the transplanted tissue. In some embodiments, tolerance comprises reducing a pathogenic immune response to a virus. In some embodiments, tolerance comprises a reduced immune response to the therapeutic agent. In some embodiments, tolerance comprises a reduced immune response to the therapeutic vehicle.

In some embodiments in which modified immune cells are administered to an individual, the method comprises at least one (such as at least 2, 3, 4, 5, 6 or more) additional administrations of the modified immune cells as described above. include additional In some embodiments, the period of time between any two administrations is at least 1 day, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or 1 year.

Certain aspects of the invention relate to systems comprising strictures, immune cells, antigens, and/or tolerogenic factors for use in any of the aforementioned methods. Certain aspects of the invention relate to a system comprising a constrictor, an immune cell, and a compound encoding a non-functional cytokine binding protein, for use in any of the aforementioned methods.

1 presents a schematic diagram of a processing schedule.
Figure 2 shows the number of OT-I- and OT-II-specific T cells at day 12 in mice from the four treatment groups.
Figure 3 shows the percentage of splenic T cells that expressed high levels of IFN-[gamma] (left) and the level of IFN-[gamma] production (right).
4 shows the number of OT-I T cells producing IFN-γ as determined by ELI spots.
5 shows representative flow cytograms for TCRVa2+ splenic OT-I and lymph node OT-II T cells for each treatment group.
6 shows representative flow cytograms for OT-I cells high in IFN-γ and CD44 antigen-activating markers.
7 presents a schematic diagram of a processing schedule.
Figure 8 shows the number of OT-I- and OT-II-specific T cells at day 12 in mice from the four treatment groups.
9 shows the percentage of splenic T cells that expressed high levels of IFN-γ.
10 shows representative flow cytograms for OT-I T cell counts versus CD8+ T cells (upper panel) and cells high in IFN-γ versus CD44 antigen-activating marker (lower panel).
11A and 11B present schematic diagrams of representative processing schedules.
12A and 12B present schematic diagrams of representative processing schedules.

The present invention provides a method for inducing tolerance and/or suppressing an immune response in a subject by passing a cell suspension containing immune cells through a stricture, thereby enabling delivery of antigens and/or tolerogenic factors to immune cells. to provide. In some embodiments, the constriction is contained within the microfluidic channel. In some embodiments, the constriction is a pore or is contained within a pore.

A particular aspect of the present disclosure is a method of inducing tolerance to an antigen and/or suppressing an immune response in a subject by passing a cell suspension comprising immune cells through a constriction, wherein the constriction modifies the cells and thereby disrupts the cells. causing the antigen to enter an immune cell, and introducing the immune cell into a subject. In some embodiments, presentation of an antigen on tolerogenic cells induces tolerance to the antigen and/or suppresses an immune response. In some embodiments, antigens are presented in a tolerogenic environment. In some embodiments, an antigen is processed in an tolerogenic environment. In some embodiments, the tolerance and/or immune suppression is antigen-specific. In some embodiments, tolerance and/or immune suppression is non-specific, comprising inhibition of tolerance and/or immune responses to multiple antigens.

A particular aspect of the present disclosure is a method of inducing tolerance to an antigen and/or suppressing an immune response in a subject by passing a cell suspension comprising immune cells through a constriction, wherein the constriction modifies the cells and thereby disrupts the cells. causing antigens and tolerogenic factors to enter immune cells, and introducing immune cells into a subject, thereby inducing tolerance to an antigen and/or suppressing an immune response. In some embodiments, the antigen is presented in an tolerogenic environment. In some embodiments, tolerogenic factors create an tolerogenic environment, and presentation of an antigen in the tolerogenic environment induces tolerance to the antigen and/or suppresses an immune response. In some embodiments, the antigen is processed in an tolerogenic environment. In some embodiments, the tolerance and/or immune suppression is antigen-specific. In some embodiments, tolerance and/or immune suppression is non-specific, comprising inhibition of tolerance and/or immune responses to multiple antigens.

A particular aspect of the present disclosure is a method of inducing tolerance to an antigen and/or suppressing an immune response in a subject by passing a cell suspension comprising immune cells through a constriction, wherein the constriction modifies the cells and thereby disrupts the cells. causing tolerogenic factors to enter immune cells, and introducing immune cells into a subject, thereby inducing tolerance to an antigen and/or suppressing an immune response. In some embodiments, tolerogenic factors create an tolerogenic environment, and presentation of an antigen in the tolerogenic environment induces tolerance to the antigen and/or suppresses an immune response. In some embodiments, the tolerance and/or immune suppression is antigen-specific. In some embodiments, tolerance and/or immune suppression is non-specific, comprising inhibition of tolerance and/or immune responses to multiple antigens.

A particular aspect of the present disclosure is a method of generating tolerogenic and/or immunosuppressive antigen-presenting cells, wherein the antigen-presenting cells pass through a constriction, wherein the constriction transforms the cells, thereby causing perturbation of the cells to allow tolerance. wherein the genic factor enters the antigen-presenting cell. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule).

I. General Skills

The techniques and procedures described or referred to herein are generally well understood and routine methodology by those skilled in the art, such as those described in Molecular Cloning: A Laboratory Manual (Sambrook et al. , 4th ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); [ Current Protocols in Molecular Biology (FM Ausubel, et al. eds., 2003)]; series [ Methods in Enzymology (Academic Press, Inc.)]; [ PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds., 1995)]; Antibodies, A Laboratory Manual ( Harlow and Lane, eds., 1988); [ Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RI Freshney, 6th ed., J. Wiley and Sons, 2010)]; [ Oligonucleotide Synthesis (MJ Gait, ed., 1984)]; [ Methods in Molecular Biology , Humana Press]; [ Cell Biology: A Laboratory Notebook (JE Cellis, ed., Academic Press, 1998)]; [ Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, Plenum Press, 1998)]; [ Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., J. Wiley and Sons, 1993-8)]; [ Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds., 1996)]; [ Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987)]; [ PCR: The Polymerase Chain Reaction , (Mullis et al. , eds., 1994)]; [ Current Protocols in Immunology (JE Coligan et al. , eds., 1991)]; [ Short Protocols in Molecular Biology (Ausubel et al. , eds., J. Wiley and Sons, 2002)]; [ Immunobiology (CA Janeway et al. , 2004)]; [ Antibodies (P. Finch, 1997)]; [ Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989)]; [ Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000)]; [ Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999)]; [ The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995)]; and Cancer: Principles and Practice of Oncology (VT DeVita et al. , eds., JB Lippincott Company, 2011).

II. Justice

For purposes of interpreting this specification, the following definitions will apply and where appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the set forth definition shall prevail.

As used herein, singular forms include plural referents unless otherwise indicated.

It is understood that aspects and embodiments of the invention described herein include "comprising", "consisting" of, and "consisting essentially of" the aspects and embodiments. .

As used herein, the term "about" refers to the typical error range for each value readily known to those skilled in the art. Reference herein to a value or parameter of “about” includes (and describes) embodiments to that value or parameter per se.

As used herein, the term "pore" refers to an opening comprising a hole, tear, cavity, gap, fracture, crevice, or perforation in a material. In some instances (where indicated) these terms refer to pores within a surface of the present disclosure. In another example, a pore (where indicated) can refer to a pore in a cell membrane.

The term "membrane" as used herein refers to a selective barrier or sheet containing pores. This term includes flexible sheet-like structures that act as borders or linings. In some instances, these terms refer to a surface or filter that includes pores. This term is distinct from the term “cell membrane”.

As used herein, the term "filter" refers to a porous article that allows selective passage through pores. In some instances, these terms refer to a surface or membrane containing pores.

The term “heterogeneous” as used herein refers to being mixed or not uniform in structure or composition. In some instances, these terms refer to pores that vary in size, shape, or distribution within a given surface.

As used herein, the term “homogeneous” refers to being consistent or uniform in structure or composition throughout. In some instances, these terms refer to pores that are consistent in size, shape, or distribution within a given surface.

As used herein, the term "heterologous" refers to molecules derived from different organisms. In some instances, these terms refer to nucleic acids or proteins that are not normally found or expressed in a given organism.

As used herein, the term “inhibit” can refer to an action that blocks, reduces, eliminates, or otherwise antagonizes the presence or activity of a particular target. Inhibition can refer to partial or complete inhibition. For example, inhibiting an immune response can refer to any action leading to blockage, reduction, elimination, or any other antagonism of an immune response. In other examples, inhibition of nucleic acid expression can include, but is not limited to, reduction of nucleic acid transcription, reduction of mRNA abundance (eg, silencing mRNA transcription), mRNA degradation, inhibition of mRNA translation, and the like. In another example, inhibition of protein expression may include, but is not limited to, reduction of transcription of a nucleic acid encoding a protein, reduction of stability of mRNA encoding a protein, inhibition of protein translation, reduction of protein stability, and the like.

As used herein, the term “inhibit” can refer to reducing, reducing, inhibiting, limiting, reducing, or otherwise reducing the presence or activity of a particular target. Inhibition can refer to partial or complete inhibition. For example, inhibiting an immune response can refer to any action that leads to reducing, lowering, inhibiting, limiting, curtailing, or otherwise reducing an immune response. In other examples, inhibition of nucleic acid expression can include, but is not limited to, reduction of nucleic acid transcription, reduction of mRNA abundance (eg, silencing mRNA transcription), mRNA degradation, inhibition of mRNA translation, and the like. In another example, inhibition of protein expression may include, but is not limited to, lowering of transcription of a nucleic acid encoding a protein, lowering of stability of mRNA encoding a protein, inhibition of protein translation, lowering of protein stability, and the like.

As used herein, the term "enhance" can refer to an action that improves, enhances, heightens, or otherwise increases the presence or activity of a particular target. For example, enhancing an immune response can refer to any action that leads to improving, enhancing, heightening, or otherwise increasing the immune response. In another example, enhancing nucleic acid expression can include, but is not limited to, increasing nucleic acid transcription, increasing mRNA abundance (eg, increasing mRNA transcription), decreasing mRNA degradation, increasing mRNA translation, and the like. don't In another example, enhancing protein expression may include, but is not limited to, increased transcription of a nucleic acid encoding the protein, increased stability of mRNA encoding the protein, increased protein translation, increased protein stability, and the like. .

As used herein, the term “induce” can refer to an action that initiates, promotes, stimulates, establishes, or otherwise produces a result. For example, inducing an immune response can refer to any action leading to initiating, promoting, stimulating, establishing, or otherwise producing a desired immune response. In another example, inducing nucleic acid expression may include, but is not limited to, initiation of nucleic acid transcription, initiation of mRNA translation, and the like. In another example, inducing protein expression may include, but is not limited to, increased transcription of a nucleic acid encoding the protein, increased stability of mRNA encoding the protein, increased protein translation, increased protein stability, and the like. .

As used herein, the term “homologous” refers to molecules derived from the same organism. In some instances, these terms refer to nucleic acids or proteins normally found or expressed in a given organism.

The term “polynucleotide” or “nucleic acid” as used herein refers to nucleotides of any length in the form of polymers, including ribonucleotides and deoxyribonucleotides. Accordingly, these terms include single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or purine and pyrimidine bases, or natural, chemically or biochemically modified, or non-natural polymers comprising other nucleotide bases that are nucleotide bases, or are derivatized, but are not limited thereto. The backbone of a polynucleotide may include sugar and phosphate groups (which may be typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. The backbone of a polynucleotide may include repeating units linked by peptide bonds, such as N-(2-aminoethyl)-glycine (ie, a peptide nucleic acid). Alternatively, the backbone of the polynucleotide may comprise polymers of synthetic subunits such as phosphoramidates, thus oligodeoxynucleoside phosphoramidates (P-NH2) or mixed phosphoramidate-phosphodisomes. It may be an ester oligomer. Additionally, double-stranded polynucleotides can be prepared from single-stranded polynucleotide products of chemical synthesis by synthesizing complementary strands and annealing the strands under suitable conditions, or by synthesizing complementary strands de novo using DNA polymerase and suitable primers. can be obtained.

The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are included in this definition. This term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Moreover, for purposes of this invention, "polypeptide" refers to a protein comprising modifications to its native sequence, such as deletions, additions and substitutions (generally conservative in nature), provided that such proteins retain the desired activity. do. Such modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as through errors due to PCR amplification or mutation of the host producing the protein.

For any structural and functional property described herein, methods for determining such property are known in the art.

III. Immune Suppression and Tolerance

In certain aspects, the invention is a method of suppressing an immune response and/or inducing tolerance in a subject, wherein a cell suspension comprising immune cells is passed through a constriction, wherein the constriction modifies the cells, thereby causing disturbance of the cells to antigenic antigens. and/or tolerogenic factors enter the immune cells, wherein tolerogenic or immunosuppressive immune cells are produced, and introducing the immune cells into the subject, wherein the presentation of the antigen to the antigen Inducing tolerance to and/or suppressing the immune response.

Tolerogenic immune cells can present antigens in an tolerogenic manner (e.g., presentation of an antigen by tolerogenic immune cells induces tolerance to the antigen) or transfer the tolerogenic phenotype to another cell, such as another antigen. -characterized as an immune cell capable of producing tolerogenic antigen-presenting cells by conferring onto presenting cells. Immunosuppressive immune cells can present antigens in an immunosuppressive manner (e.g., presentation of antigens by immunosuppressive immune cells suppresses the immune response to the antigens) or transfers an immunosuppressive phenotype to another. It is characterized as an immune cell capable of generating an immunosuppressive antigen-presenting cell by conferring onto a cell, such as another antigen-presenting cell.

In some embodiments, tolerogenic and/or immunosuppressive immune cells, or precursors thereof, described herein have not been contacted with an adjuvant. In some embodiments, the adjuvant activates pattern recognition receptor receptors (PRRs) (e.g., toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and C-type lectins). receptors (CLR)), and pathogen-associated molecular patterns (PAMPs). In some embodiments, the adjuvant is a TLR3 and RLR ligand (e.g., a synthetic analog of dsRNA, such as poly(I:C)), a TLR4 ligand (e.g., lipopolysaccharide (LPS), monophosphoryl lipid A ( MPLA)), TLR5 ligands (e.g., bacterial flagellin (alone or fused to an antigen)), TLR7/8 ligands (e.g., imidazoquinolines such as imiquimod, gardiquimod, and R848), TLR9 ligands (e.g. CpG ODNs such as oligodeoxynucleotides containing specific CpG motifs, including ODN 1826 and ODN 2006), NOD2 ligands (e.g. bacterial cell wall fragments such as muramyl dipeptide (MDP)) , alum, water-in-oil emulsion (Freund's incomplete adjuvant, MF59), rhIL-2, anti-CD40 or CD40L, IL-12, and cyclic dinucleotides.

In some embodiments, a tolerogenic and/or immunosuppressive immune cell described herein comprises a reduced ability to provide one or more costimulatory signals when compared to a non-tolerogenic and/or non-immunosuppressive precursor. In some embodiments, a costimulatory signal comprises signaling through a CD40, CD80, CD86, CD54, CD83, CD79, or ICOS ligand.

In some embodiments, tolerogenic and/or immunosuppressive immune cells described herein comprise a reduced ability to provide one or more inflammatory signals when compared to a non-tolerogenic and/or non-immunosuppressive precursor. In some embodiments, the inflammatory signal is interleukin-1 (IL-1), IL-12, IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma), granulocyte-macrophage colony stimulating factor (GM- CSF), NF-κB, interferon regulatory factor (IRF), or JAK-STAT.

Immunological tolerance is an immunological unresponsiveness to challenge with an antigen for which tolerance has been induced. Tolerance is demonstrated when the subject is unresponsive to a subsequent challenge with a tolerance-inducing antigen. Tolerance can be mediated by several different mechanisms including apoptosis of autoreactive cells, induction of anergy, or bias in the lymphocyte phenotype. In some embodiments, tolerance may include T cell non-reactivity resulting in inactivation of an immune response to a particular antigen. Tolerance may also be mediated by regulatory T cells (Tregs) by contact-dependent interactions with cell surface molecules or cytotoxic factors or through secretion of immunosuppressive cytokines. Defects in tolerogenic mechanisms can contribute to autoimmune diseases, transplant rejection, anti-drug responses, and pathogenic responses to viral infections. Immunosuppression is a decrease in the activity of an immune response. For example, immunosuppression can include, but is not limited to, reducing responsiveness to antigenic challenge, reducing immune cell activation and proliferation, modulating cytokine production and/or secretion, reducing immune cell survival, or reducing immune cell effector function. .

In some embodiments, the invention provides a method of delivering to an immune cell, wherein the cell is a mammalian cell. In some embodiments, the cell is a primary cell or cell line cell. In some embodiments, the cell is a blood cell. In some embodiments, the blood cells are immune cells. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the immune cell is a T cell, B cell, natural killer (NK) cell, dendritic cell (DC), NKT cell, mast cell, monocyte, macrophage, basophil, eosinophil, or neutrophil. In some embodiments, the immune cell is an adoptive immune cell such as a T cell or B cell. In some embodiments, an immune cell is an extracellularly generated cell (eg, an extracellularly generated dendritic cell). Exemplary extracellularly generated cells include immune cells generated from stem cells or precursor cells (eg, autologous stem cells or precursor cells). In some embodiments, the immune cell is an innate immune cell. Exemplary innate immune cells include innate lymphoid cells (ILC 1, ILC2, ILC3), basophils, eosinophils, mast cells, NK cells, neutrophils, and monocytes. In some embodiments, the immune cells are memory cells. In some embodiments, the immune cell is a primary human T cell. In some embodiments, the cell is a mouse, dog, cat, horse, rat, goat, monkey, or rabbit cell. In some embodiments, the cell is a human cell. In some embodiments, a cell suspension comprises non-mammalian cells. In some embodiments, the cell is a chicken, frog, insect, fish or nematode cell.

In certain aspects, the present invention is a method of inducing tolerance to an antigen in a subject by passing a cell suspension comprising immune cells through a constriction, wherein the constriction transforms the cells, causing disturbance of the cells, thereby causing antigen contact with the cells. entering the immune cell, and introducing the immune cell into a subject, wherein the immune cell is a tolerogenic immune cell, and wherein the presentation of the antigen induces tolerance to the antigen. to provide. In some embodiments, an antigen is presented by tolerogenic immune cells to induce tolerance to the antigen. In some embodiments, tolerogenic immune cells confer an tolerogenic phenotype on another antigen-presenting cell, thereby generating tolerogenic antigen-presenting cells, and antigens are presented by tolerogenic antigen-presenting cells to antigen induce tolerance for In some embodiments, tolerance is antigen-specific.

In certain aspects, the present invention is a method of inducing tolerance to an antigen in a subject by passing a cell suspension comprising immune cells through a constriction, wherein the constriction modifies the cells, causing disturbance of the cells, thereby causing antigen contact with the cells. and wherein the tolerogenic factor enters the immune cells, thereby generating tolerogenic immune cells comprising the antigen, and introducing the tolerogenic immune cells into the subject, wherein the presentation of the antigen is It provides a method of inducing tolerance for. In some embodiments, an antigen is presented by tolerogenic immune cells to induce tolerance to the antigen. In some embodiments, tolerogenic immune cells confer an tolerogenic phenotype onto another antigen-presenting cell, thereby generating tolerogenic antigen-presenting cells, and antigens are presented by tolerogenic antigen-presenting cells to antigen induce tolerance for In some embodiments, tolerance is antigen-specific.

In certain aspects, the invention is a method of suppressing an immune response to an antigen in a subject, wherein a cell suspension comprising immune cells is passed through a constriction, wherein the constriction modifies the cells, thereby causing disturbance of the cells, thereby contacting the cells. wherein the antigen enters an immune cell, and introduces an immunosuppressive immune cell into the subject, wherein the immune cell is an immunosuppressive immune cell, wherein the presentation of the antigen elicits an immune response to the antigen. It provides a way to suppress. In some embodiments, an antigen is presented by immunosuppressive immune cells to suppress an immune response to the antigen. In some embodiments, an immunosuppressive immune cell confers an immunosuppressive phenotype on another antigen-presenting cell, thereby generating an immunosuppressive antigen-presenting cell, and is induced by the immunosuppressive antigen-presenting cell to antigen It is presented and suppresses the immune response to the antigen. In some embodiments, immunosuppression is antigen-specific.

In certain aspects, the invention is a method of suppressing an immune response to an antigen in a subject, wherein a cell suspension comprising immune cells is passed through a constriction, wherein the constriction modifies the cells, thereby causing disturbance of the cells, thereby contacting the cells. wherein the antigen and tolerogenic factor enter the immune cell, thereby generating immunosuppressive immune cells comprising the antigen, and introducing the immunosuppressive immune cell into the subject, wherein the antigen The presentation is to suppress an immune response to the antigen. In some embodiments, an antigen is presented by immunosuppressive immune cells to suppress an immune response to the antigen. In some embodiments, an immunosuppressive immune cell confers an immunosuppressive phenotype on another antigen-presenting cell, thereby generating an immunosuppressive antigen-presenting cell, and is induced by the immunosuppressive antigen-presenting cell to antigen It is presented and suppresses the immune response to the antigen. In some embodiments, immunosuppression is antigen-specific.

In certain aspects, the invention is a method of inducing tolerance to an antigen in a subject comprising introducing tolerogenic immune cells into the subject, wherein the tolerogenic immune cells comprise an antigen, wherein the presentation of the antigen comprises the antigen. induces tolerance to, wherein the antigen has been introduced into tolerogenic immune cells by passing the tolerogenic immune cells or precursors thereof to the constriction, wherein the constriction transforms the cells, causing disturbance of the cells, so that the antigen is introduced into the tolerogenic immune cells. It provides a method that has entered the cell or its precursor. In some embodiments, an antigen is presented by tolerogenic immune cells to induce tolerance to the antigen. In some embodiments, tolerogenic immune cells confer an tolerogenic phenotype on another antigen-presenting cell, thereby generating tolerogenic antigen-presenting cells, and antigens are presented by tolerogenic antigen-presenting cells to antigen induce tolerance for In some embodiments, tolerance is antigen-specific.

In certain aspects, the invention is a method of inducing tolerance to an antigen in a subject comprising introducing tolerogenic immune cells into the subject, wherein the tolerogenic immune cells comprise an antigen and tolerogenic factors, wherein the Presentation of the antigen induces tolerance to the antigen, wherein the antigen and tolerogenic factors have been introduced into the tolerogenic immune cells by passing the tolerogenic immune cells or their precursors to a stricture, wherein the stricture transforms the cell so that the cell It provides a method wherein antigens and tolerogenic factors enter tolerogenic immune cells or precursors thereof by causing perturbation of. In some embodiments, an antigen is presented by tolerogenic immune cells to induce tolerance to the antigen. In some embodiments, tolerogenic immune cells confer an tolerogenic phenotype onto another antigen-presenting cell, thereby generating tolerogenic antigen-presenting cells, and antigens are presented by tolerogenic antigen-presenting cells to antigen induce tolerance for In some embodiments, tolerance is antigen-specific.

In certain aspects, the invention is a method of suppressing an immune response to an antigen in a subject comprising introducing immunosuppressive immune cells into the subject, wherein the immunosuppressive immune cells comprise the antigen, wherein the antigen is Presentation inhibits an immune response to an antigen, wherein the antigen has been introduced to the immunosuppressive immune cells by passing the immunosuppressive immune cells or their precursors to a constriction, wherein the constriction modifies the cell and thereby disrupts the cell. causing the antigen to enter immunosuppressive immune cells or precursors thereof. In some embodiments, an antigen is presented by immunosuppressive immune cells to suppress an immune response to the antigen. In some embodiments, an immunosuppressive immune cell confers an immunosuppressive phenotype on another antigen-presenting cell, thereby generating an immunosuppressive antigen-presenting cell, and is induced by the immunosuppressive antigen-presenting cell to antigen It is presented and suppresses the immune response to the antigen. In some embodiments, immunosuppression is antigen-specific.

In certain aspects, the invention is a method of suppressing an immune response to an antigen in a subject comprising introducing immunosuppressive immune cells into the subject, wherein the immunosuppressive immune cells comprise an antigen and tolerogenic factors; wherein the presentation of the antigen suppresses an immune response to the antigen, wherein the antigen and tolerogenic factor have been introduced into the immunosuppressive immune cell by passing the immunosuppressive immune cell or a precursor thereof to a stricture, wherein the stricture blocks the cell It provides a method wherein the cell is disrupted by transformation so that antigens and tolerogenic factors enter immunosuppressive immune cells or precursors thereof. In some embodiments, an antigen is presented by immunosuppressive immune cells to suppress an immune response to the antigen. In some embodiments, an immunosuppressive immune cell confers an immunosuppressive phenotype on another antigen-presenting cell, thereby generating an immunosuppressive antigen-presenting cell, and is induced by the immunosuppressive antigen-presenting cell to antigen It is presented and suppresses the immune response to the antigen. In some embodiments, immunosuppression is antigen-specific.

In certain aspects, the present invention is a method of generating tolerogenic immune cells, comprising passing a cell suspension comprising immune cells through a constriction, wherein the constriction modifies the cells, thereby causing disturbance of the cells, thereby contacting the cells. Tolerogenic factors enter immune cells, thereby generating tolerogenic immune cells. In certain aspects, the invention is a method of generating tolerogenic antigen-presenting immune cells, wherein the tolerogenic immune cells further pass through a second constriction, wherein the second constriction causes disruption of the cells by modifying the cells, An antigen contacted with a cell enters an immune cell, wherein the antigen is presented by a tolerogenic immune cell.

In certain aspects, the invention is a method of generating immunosuppressive immune cells, comprising passing a cell suspension comprising immune cells through a constriction, wherein the constriction deforms the cells, thereby causing disturbance of the cells to contact the cells. Provided is a method wherein the tolerogenic factor entered into immune cells, thereby generating immunosuppressive immune cells. In certain aspects, the invention is a method of generating immunosuppressive antigen-presenting immune cells, wherein the immunosuppressive immune cells further pass through a second constriction, wherein the second constriction modifies the cells, thereby disrupting the cells. to cause antigens contacted with cells to enter immune cells, wherein the antigens are presented by immunosuppressive immune cells.

In some embodiments, immune suppression and/or tolerance comprises modulation of the expression and/or activity of an immunomodulatory agent (eg, a costimulatory molecule, immunosuppressive agent, or inflammatory or anti-inflammatory molecule). In some embodiments, immune suppression and/or tolerance comprises inhibiting the expression and/or activity of an immunostimulatory agent (e.g., a co-stimulatory molecule), enhancing the expression and/or activity of an immunosuppressive agent, expressing and/or expressing an inflammatory molecule, and/or inhibition of activity, and/or enhancement of expression and/or activity of anti-inflammatory molecules.

In some embodiments, immune suppression and/or tolerance comprises inhibition of the expression and/or activity of an immunostimulatory agent (eg, costimulatory molecule). In some embodiments, an immunostimulatory agent is a costimulatory molecule. Interactions between costimulatory molecules and their ligands are important for sustaining and integrating TCR signaling to stimulate optimal T cell proliferation and differentiation. In some embodiments, immune suppression and/or tolerance comprises reducing expression of costimulatory molecules. Exemplary costimulatory molecules expressed on antigen-presenting cells include, but are not limited to, CD40, CD80, CD86, CD54, CD83, CD79, or ICOS ligands. In some embodiments, the costimulatory molecule is CD80 or CD86. In some embodiments, immune suppression and/or tolerance comprises inhibition of expression of a nucleic acid that expresses or modulates the expression of a costimulatory molecule. In some embodiments, a nucleic acid that expresses or modulates expression of a costimulatory molecule is deleted. In some embodiments, deletion of a nucleic acid that expresses or modulates the expression of a costimulatory molecule is achieved through gene editing. In some embodiments, immune suppression and/or tolerance comprises inhibition of costimulatory molecules. In some embodiments, immune suppression and/or tolerance comprises inhibition of the expression or function of costimulatory molecules. In some embodiments, immune suppression and/or tolerance comprises increasing the activity of transcriptional regulators that inhibit expression of costimulatory molecules. In some embodiments, immune suppression and/or tolerance comprises increasing the activity of a protein inhibitor that inhibits expression of costimulatory molecules. In some embodiments, immune suppression and/or tolerance comprises degradation of costimulatory molecules. In some embodiments, immune suppression and/or tolerance comprises labeling costimulatory molecules for destruction. For example, immune suppression and/or tolerance can include targeting costimulatory molecules for destruction by ubiquitination.

In some embodiments, immune suppression and/or tolerance comprises enhancing the expression and/or activity of an immunosuppressive agent. In some embodiments, the immunosuppressive agent is a co-inhibitory molecule, transcriptional regulator, or immunosuppressive molecule. Co-inhibitory molecules negatively regulate lymphocyte activation. Exemplary co-inhibitory molecules include, but are not limited to, PD-L1, PD-L2, HVEM, B7-H3, TRAIL, immunoglobulin-like transcript (ILT) receptors (ILT2, ILT3, ILT4), FasL, CTLA4, CD39, CD73, and B7-H4. In some embodiments, the co-inhibitory molecule is PD-L1 or PD-L2. In some embodiments, immune suppression and/or tolerance comprises increasing the activity of co-inhibitory molecules. In some embodiments, immune suppression and/or tolerance comprises increased expression of co-inhibitory molecules. In some embodiments, immune suppression and/or tolerance comprises increasing the activity of transcriptional regulators that enhance expression of co-inhibitory molecules. In some embodiments, immune suppression and/or tolerance comprises increasing the activity of a polypeptide that increases expression of a co-inhibitory molecule. In some embodiments, immune suppression and/or tolerance comprises inhibition of an inhibitor of a co-inhibitory molecule.

In some embodiments, immune suppression and/or tolerance comprises increasing the expression and/or activity of an immunosuppressive molecule. Exemplary immunosuppressive molecules include, but are not limited to, arginase-1 (ARG1), indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), Nitric oxide (NO), nitric oxide synthase 2 (NOS2), TSLP, vascular intestinal peptide (VIP), hepatocyte growth factor (HGF), TGFβ, IFNα, IL-4, IL-10, IL-13, and IL- contains 35 In some embodiments, the immunosuppressive molecule is NO or IDO. In some embodiments, immune suppression and/or tolerance comprises increasing the activity of an immunosuppressive molecule. In some embodiments, immune suppression and/or tolerance comprises increasing the activity of transcriptional regulators that enhance expression of immunosuppressive molecules. In some embodiments, immune suppression and/or tolerance comprises increasing the activity of a polypeptide that enhances the expression of an immunosuppressive molecule. In some embodiments, immune suppression and/or tolerance comprises inhibition of negative regulators of immunosuppressive molecules.

In some embodiments, immune suppression and/or tolerance comprises inhibition of the expression and/or activity of inflammatory molecules. In some embodiments, the inflammatory molecule is an inflammatory cytokine. In some embodiments, the inflammatory cytokines are interleukin-1 (IL-1), IL-12, and IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma), and granulocyte-macrophage colony stimulating factor ( GM-CSF). In some embodiments, the inflammatory molecule is an inflammatory transcription factor. In some embodiments, immune suppression and/or tolerance comprises inhibition of inflammatory transcription factors. In some embodiments, immune suppression and/or tolerance comprises reducing expression of inflammatory transcription factors. In some embodiments, the inflammatory transcription factor is a molecule associated with NF-κB, interferon regulatory factor (IRF), or JAK-STAT signaling pathway. The NF-κB pathway is a pro-inflammatory signaling pathway that mediates the expression of pro-inflammatory genes including cytokines, chemokines and adhesion molecules. Interferon regulatory factors (IRFs) constitute a family of transcription factors that can regulate the expression of pro-inflammatory genes. The JAK-STAT signaling pathway transmits information from extracellular cytokine signals to the nucleus, resulting in DNA transcription and expression of genes involved in immune cell proliferation and differentiation. The JAK-STAT system consists of a cell surface receptor, Janus kinase (JAK), and signal transducer and activator of transcription (STAT) proteins. Exemplary JAK-STAT molecules include, but are not limited to, JAK1, JAK2, JAK 3, Tyk2, STAT1, STAT2, STAT3, STAT4, STAT5 (STAT5A and STAT5B), and STAT6. In some embodiments, immune suppression and/or tolerance comprises enhancing expression of an inhibitor of cytokine signaling (SOCS) protein. SOCS proteins can inhibit signaling through the JAK-STAT pathway. In some embodiments, immune suppression and/or tolerance comprises inhibition of expression of a nucleic acid encoding an inflammatory transcription factor. In some embodiments, a nucleic acid encoding an inflammatory transcription factor is deleted. In some embodiments, immune suppression and/or tolerance comprises increasing the activity of transcriptional regulators that inhibit expression of inflammatory transcription factors. In some embodiments, immune suppression and/or tolerance comprises increasing the activity of a protein inhibitor that inhibits the expression of inflammatory transcription factors.

In some embodiments, immune suppression and/or tolerance comprises enhancing the expression and/or activity of anti-inflammatory molecules. In some embodiments, the anti-inflammatory molecule is an anti-inflammatory cytokine. In some embodiments, the anti-inflammatory cytokine is selected from IL-4, IL-10, IL-13, IFN-alpha and transforming growth factor-beta (TGFβ). In some embodiments, the anti-inflammatory molecule is an anti-inflammatory transcription factor. In some embodiments, immune suppression and/or tolerance comprises enhancement of anti-inflammatory transcription factors. In some embodiments, immune suppression and/or tolerance comprises increased expression of anti-inflammatory transcription factors. In some embodiments, immune suppression and/or tolerance comprises enhancing expression of nucleic acids encoding anti-inflammatory transcription factors. In some embodiments, a nucleic acid encoding an anti-inflammatory transcription factor is deleted. In some embodiments, immune suppression and/or tolerance comprises reducing the activity of transcriptional regulators that inhibit expression of anti-inflammatory transcription factors. In some embodiments, immune suppression and/or tolerance comprises reducing the activity of a protein inhibitor that inhibits expression of an anti-inflammatory transcription factor.

In certain aspects, the invention is a method of generating a tolerogenic antigen-presenting cell comprising an antigen, wherein the tolerogenic antigen-presenting cell passes through a constriction, wherein the constriction transforms the cell, causing perturbation of the cell, thereby generating an antigen entering the tolerogenic antigen-presenting cells, thereby generating tolerogenic antigen-presenting cells comprising the antigen.

In certain aspects, the invention is a method of generating tolerogenic antigen-presenting cells, wherein the antigen-presenting cells pass through a constriction, wherein the constriction transforms the cells, causing perturbation of the cells so that the tolerogenic factor is antigen-presenting. and enters cells, thereby generating tolerogenic antigen-presenting cells. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule).

In certain aspects, the invention is a method of generating tolerogenic antigen-presenting cells comprising an antigen, wherein the antigen-presenting cells pass through a constriction, wherein the constriction transforms the cells, thereby causing disruption of the cells, thereby allowing antigen and tolerance wherein the genic factor enters the antigen-presenting cell, thereby generating tolerogenic antigen-presenting cells comprising the antigen. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule).

In certain aspects, the invention is a method of generating an immunosuppressive antigen-presenting cell comprising an antigen, wherein the immunosuppressive antigen-presenting cell passes through a constriction, wherein the constriction modifies the cell, thereby preventing perturbation of the cell. causing the antigen to enter the immunosuppressive antigen-presenting cell, thereby generating an immunosuppressive antigen-presenting cell comprising the antigen.

In certain aspects, the invention is a method of generating immunosuppressive antigen-presenting cells, wherein the antigen-presenting cells pass through a constriction, wherein the constriction transforms the cells, thereby causing perturbation of the cells so that the tolerogenic factor is antigen-presenting. and enters presenting cells, thereby generating immunosuppressive antigen-presenting cells. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule).

In certain aspects, the invention is a method of generating an immunosuppressive antigen-presenting cell comprising an antigen, wherein the antigen-presenting cell passes through a constriction, wherein the constriction transforms the cell, causing perturbation of the cell, thereby generating antigen and wherein the tolerogenic factor enters the antigen-presenting cell, thereby generating an immunosuppressive antigen-presenting cell comprising the antigen. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule).

In certain aspects, the invention is a method of delivering an antigen into first immune cells (eg, tolerogenic immune cells) comprising passing a cell suspension comprising the first immune cells through a stricture, wherein the stricture provides a method wherein a cell suspension is contacted with an antigen by modifying a first immune cell to cause disruption of the cell so that an antigen enters the cell. In some embodiments, a tolerogenic factor is delivered to a first immune cell by a method disclosed herein, thereby conferring a tolerogenic phenotype to the first immune cell. In some embodiments, the antigen is presented in a tolerogenic manner, eg, by a first immune cell. In some embodiments, the antigen is another immune cell, e.g., a second immune cell, such as an immune cell having an tolerogenic phenotype conferred by the nature of the first immune cell, or tolerogenicity independent of the first immune cell. It is presented in a tolerogenic manner by immune cells with a phenotype. In some embodiments, antigens are processed by tolerogenic immune cells (eg, by first or second immune cells) and presented by class I MHCs. In some embodiments, antigens are processed by tolerogenic immune cells (eg, by first or second immune cells) and presented by class II MHCs.

In certain aspects, the invention is a method of delivering a tolerogenic factor that produces a tolerogenic phenotype into first immune cells, comprising passing a cell suspension comprising the first immune cells through a constriction, wherein the constriction is immune to immune cells. Transforming the cells causes perturbation of the cells so that tolerogenic factors enter the cells, wherein the cell suspension is contacted with the tolerogenic factors, thereby generating tolerogenic immune cells. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule). In some embodiments, the antigen is endocytosed by a first immune cell or tolerogenic immune cell. In some embodiments, an antigen is delivered to a first immune cell or tolerogenic immune cell by any of the methods disclosed herein. In some embodiments, antigens are presented in a tolerogenic manner, eg, by tolerogenic immune cells. In some embodiments, the antigen is another immune cell, e.g., an immune cell having a tolerogenic phenotype conferred by the properties of a second immune cell, e.g., a tolerogenic immune cell, or independent of a tolerogenic immune cell. It is presented in a tolerogenic manner by immune cells with a tolerogenic phenotype. In some embodiments, the antigen is processed by tolerogenic immune cells (eg, by tolerogenic immune cells or secondary immune cells) and presented by class I MHCs. In some embodiments, the antigen is processed by tolerogenic immune cells (eg, by tolerogenic immune cells or secondary immune cells) and presented by class II MHC.

In certain aspects, the invention is a method of delivering antigens and tolerogenic factors that produce an tolerogenic phenotype into first immune cells, comprising passing a cell suspension comprising the first immune cells through a stricture, wherein the stricture transforms immune cells to cause cell perturbation so that antigens and tolerogenic factors enter the cells, wherein the cell suspension is contacted with the antigens and tolerogenic factors, thereby generating tolerogenic immune cells comprising the antigens. provides In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule). In some embodiments, antigens are presented in a tolerogenic manner, eg, by tolerogenic immune cells. In some embodiments, the antigen is independent of another immune cell, e.g., a second immune cell, e.g., an immune cell having a tolerogenic phenotype conferred by the properties of a tolerogenic immune cell, or a tolerogenic immune cell. It is presented in a tolerogenic manner by immune cells with a phosphorus tolerogenic phenotype. In some embodiments, the antigen is processed by tolerogenic immune cells (eg, by tolerogenic immune cells or secondary immune cells) and presented by class I MHCs. In some embodiments, the antigen is processed by tolerogenic immune cells (eg, by tolerogenic immune cells or secondary immune cells) and presented by class II MHC.

In certain aspects, the invention is a method of delivering an antigen into first immune cells (eg, immunosuppressive immune cells), comprising passing a cell suspension comprising the first immune cells through a constriction, wherein: The constriction modifies the first immune cell to cause perturbation of the cell so that the antigen enters the cell, wherein the cell suspension is contacted with the antigen. In some embodiments, the tolerogenic factor is delivered to the first immune cell by a method disclosed herein, thereby conferring an immunosuppressive phenotype to the first immune cell. In some embodiments, the antigen is presented in an immunosuppressive manner, eg, by a first immune cell. In some embodiments, the antigen is another immune cell, e.g., a second immune cell, such as an immune cell with an immunosuppressive phenotype conferred by the nature of the first immune cell, or an immune cell independent of the first immune cell. It is presented in an immunosuppressive manner by immune cells with a suppressive phenotype. In some embodiments, the antigen is processed by an immunosuppressive immune cell (eg, by a first or second immune cell) and presented by a class I MHC. In some embodiments, the antigen is processed by an immunosuppressive immune cell (eg, by a first or second immune cell) and presented by a class II MHC.

In certain aspects, the invention is a method of delivering tolerogenic factors that produce an immunosuppressive phenotype into immune cells, comprising passing a cell suspension comprising immune cells through a constriction, wherein the constriction modifies the immune cells. thereby causing perturbation of the cells so that tolerogenic factors that produce an immunosuppressive phenotype enter the cells, wherein the cell suspension is contacted with the tolerogenic factors, thereby generating immunosuppressive immune cells. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule). In some embodiments, the antigen is endocytosed by a first immune cell or an immunosuppressive immune cell. In some embodiments, an antigen is delivered to a first immune cell or an immunosuppressive immune cell by any of the methods disclosed herein. In some embodiments, antigens are presented in an immunosuppressive manner, eg, by immunosuppressive immune cells. In some embodiments, the antigen is another immune cell, e.g., a second immune cell, e.g., an immune cell with an immunosuppressive phenotype conferred by the nature of an immunosuppressive immune cell, or an immunosuppressive immune cell. It is presented in an immunosuppressive manner by immune cells with a cell-independent tolerogenic phenotype. In some embodiments, the antigen is processed by an immunosuppressive immune cell (eg, by an immunosuppressive immune cell or a second immune cell) and presented by a class I MHC. In some embodiments, the antigen is processed by an immunosuppressive immune cell (eg, by an immunosuppressive immune cell or a second immune cell) and presented by a class II MHC.

In certain aspects, the invention is a method of delivering antigens and tolerogenic factors that produce an immunosuppressive phenotype into first immune cells, comprising passing a cell suspension comprising the first immune cells through a constriction, wherein: The constriction modifies the immune cells, causing cellular disruption so that antigens and tolerogenic factors enter the cells, where the cell suspension is contacted with the antigens and tolerogenic factors, thereby generating immunosuppressive immune cells containing the antigens. provides a way to do it. In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule). In some embodiments, antigens are presented in an immunosuppressive manner, eg, by immunosuppressive immune cells. In some embodiments, the antigen is another immune cell, e.g., a second immune cell, e.g., an immune cell with an immunosuppressive phenotype conferred by the nature of an immunosuppressive immune cell, or an immunosuppressive immune cell. It is presented in an immunosuppressive manner by immune cells with a cell-independent immunosuppressive phenotype. In some embodiments, the antigen is processed by an immunosuppressive immune cell (eg, by an immunosuppressive immune cell or a second immune cell) and presented by a class I MHC. In some embodiments, the antigen is processed by an immunosuppressive immune cell (eg, by an immunosuppressive immune cell or a second immune cell) and presented by a class II MHC.

In certain aspects, the invention is a method of suppressing an immune response in a subject, wherein a first cell suspension comprising a first immune cell is passed through a constriction, wherein the constriction modifies the cells, thereby causing disruption of the cells so that antigens are immune. entering the cell, passing a second cell suspension comprising a second immune cell through the constriction, wherein the constriction causes disturbance of the cell by transforming the cell so that the tolerogenic factor enters the immune cell, wherein the immune cell suppressive immune cells are produced, and introducing the first and second immune cells into the subject, wherein the antigen is presented in an immunosuppressive manner and suppresses the immune response to the antigen. provides a way to In some embodiments, the antigen is presented on a first immune cell. In some embodiments, the first immune cell has a tolerogenic phenotype conferred by the nature of the second immune cell. In some embodiments, antigens are presented by secondary immune cells. In some embodiments, the antigen is presented by a third immune cell, eg, an immune cell that has a tolerogenic phenotype conferred by the nature of the second immune cell. In some embodiments, the third immune cell has an tolerogenic phenotype independent of the nature of the second immune cell (e.g., the third immune cell had an tolerogenic phenotype prior to introduction of the second immune cell into the individual). . In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule). In some embodiments, the first immune cells and the second immune cells are introduced simultaneously. In some embodiments, the first immune cells and the second immune cells are combined in a cell suspension prior to introduction into a subject. In some embodiments, the first immune cells and the second immune cells are introduced sequentially. In some embodiments, a first immune cell is introduced into an individual followed by a second immune cell. In some embodiments, about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours after the first immune cells are introduced into the individual. After an hour or more than any of 24 hours, a second immune cell is introduced. In some embodiments, the first immune cells are introduced after the second immune cells are introduced into the individual. In some embodiments, about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours after the second immune cells are introduced into the individual. After an hour or more than any of 24 hours, the first immune cells are introduced. In some embodiments, delivering the tolerogenic factor to the second cell confers a secretory function on the second cell. For example, the second cell may enhance the immunosuppressive microenvironment by secreting IL-4, IL-10, IL-13, IFNα and/or TGFβ. In some embodiments, the immunosuppressive microenvironment permits tolerogenic presentation of antigen by the first cell.

In certain aspects, the invention is a method of inducing antigen-specific tolerance in a subject, wherein a first cell suspension comprising a first immune cell is passed through a constriction, wherein the constriction causes disruption of the cells by modifying the cells, wherein the antigen enters the immune cells, a second cell suspension comprising second immune cells is passed through the constriction, wherein the constriction causes disturbance of the cells by transforming the cells so that the tolerogenic factor enters the immune cells. and introducing a first immune cell and a second immune cell into a subject, wherein the antigen is presented in a tolerogenic manner and suppresses an immune response to the antigen. In some embodiments, the antigen is presented on a first immune cell. In some embodiments, the first immune cell has a tolerogenic phenotype conferred by the nature of the second immune cell. In some embodiments, antigens are presented by secondary immune cells. In some embodiments, the antigen is presented by a third immune cell, eg, an immune cell that has a tolerogenic phenotype conferred by the nature of the second immune cell. In some embodiments, the third immune cell has an tolerogenic phenotype independent of the nature of the second immune cell (e.g., the third immune cell had an tolerogenic phenotype prior to introduction of the second immune cell into the individual). . In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule). In some embodiments, the first immune cells and the second immune cells are introduced simultaneously. In some embodiments, the first immune cells and the second immune cells are introduced sequentially. In some embodiments, the first immune cells and the second immune cells are combined in a cell suspension prior to introduction into a subject. In some embodiments, a first immune cell is introduced into an individual followed by a second immune cell. In some embodiments, about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours after the first immune cells are introduced into the individual. After an hour or more than any of 24 hours, a second immune cell is introduced. In some embodiments, the first immune cells are introduced after the second immune cells are introduced into the individual. In some embodiments, about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours after the second immune cells are introduced into the individual. After an hour or more than any of 24 hours, the first immune cells are introduced. In some embodiments, delivering the tolerogenic factor to the second cell confers a secretory function on the second cell. For example, the second cell may enhance the immunosuppressive microenvironment by secreting IL-4, IL-10, IL-13, IFNα and/or TGFβ. In some embodiments, the immunosuppressive microenvironment permits tolerogenic presentation of antigen by the first cell.

In certain aspects, the invention is a method of suppressing an immune response in a subject by passing a cell suspension comprising immune cells through a constriction, wherein the constriction modifies the cells to cause perturbation of the cells to release a non-functional cytokine binding protein. wherein the encoding compound enters an immune cell, and introducing the immune cell into a subject, wherein the non-functional cytokine binding protein is expressed, wherein the non-functional cytokine binding protein is a free inflammatory cytokine It binds to, thereby providing a method of suppressing the immune response. For example, non-functional cytokine binding proteins can bind to and/or sequester free inflammatory cytokines, thereby preventing these cytokines from binding to functional cytokine receptors and triggering downstream signaling. In some embodiments, the cytokine is destroyed upon internalization and intracellular processing of the non-functional cytokine binding protein. In some embodiments, the non-functional cytokine binding protein comprises a non-functional cytokine receptor. In some embodiments, the non-functional cytokine receptor lacks a cytoplasmic signaling domain. In some embodiments, the non-functional cytokine binding protein comprises a proteolytic site that cleaves a target cytokine. For example, a non-functional cytokine binding protein may contain enzymes that cleave or degrade the cytokine upon binding. In some embodiments, the non-functional cytokine binding protein comprises an anti-cytokine antibody. In some embodiments, the non-functional cytokine binding protein comprises a B cell receptor. Exemplary cytokines to which non-functional cytokine receptors can bind include, but are not limited to, IL-2, IL-6, TNFα, IL-1, and IFNγ.

In some embodiments, at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, The immune response is suppressed by any of about 90%, or about 100%. In some embodiments, suppressing an immune response and/or inducing tolerance comprises reducing a T cell response. For example, a decrease in T cell response can include, but is not limited to, reduced T cell activation or proliferation, reduced T cell survival, or reduced cell functionality. In some embodiments, reducing a T cell response comprises reducing T cell activation. In some embodiments, reducing a T cell response comprises reducing T cell survival. In some embodiments, reducing a T cell response comprises reducing T cell proliferation. In some embodiments, reducing a T cell response comprises reducing T cell functionality. For example, reduced T cell functionality can include, but are not limited to, modulating cytokine secretion, reducing T cell migration to sites of inflammation, and reducing T cell cytotoxic activity. In some embodiments, suppressing an immune response and/or inducing tolerance comprises reducing inflammatory cytokine production and/or secretion, and/or increasing anti-inflammatory cytokine production and/or secretion. In some embodiments, suppressing an immune response and/or inducing tolerance comprises interleukin-1 (IL-1), IL-12, and IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma), and granulocyte- and reducing the production and/or secretion of one or more inflammatory cytokines selected from macrophage colony stimulating factor (GM-CSF). In some embodiments, the suppression of an immune response and/or induction of tolerance involves one or more anti-inflammatory cytokines selected from IL-4, IL-10, IL-13, IL-35, IFN-alpha, and transforming growth factor-beta (TGFβ). reduction in the production and/or secretion of In some embodiments, suppressing an immune response and/or inducing tolerance comprises a change in a T cell phenotype. For example, the T cell state may change from a pro-inflammatory phenotype to a regulatory or anti-inflammatory phenotype. In some embodiments, suppressing an immune response and/or inducing tolerance comprises non-co-stimulatory activation of T cells, which may subsequently lead to cell death. In some embodiments, suppressing an immune response and/or inducing tolerance comprises enhancing a regulatory T cell (Treg) response. In some embodiments, suppressing an immune response and/or inducing tolerance comprises enhancing a regulatory B cell (Breg), tolerogenic monocyte, bone marrow derived suppressor cell, or tolerogenic macrophage response. In some embodiments, suppressing an immune response and/or inducing tolerance comprises reducing a B cell response. In some embodiments, reducing a B cell response comprises reducing antibody production.

In some embodiments, suppressing an immune response and/or inducing tolerance comprises reducing an autoimmune response. For example, reducing an autoimmune response can include, but is not limited to, reducing an immune response to or inducing tolerance to antigens associated with type I diabetes, rheumatoid arthritis, psoriasis, multiple sclerosis, Crohn's disease, or ulcerative colitis. there is. In some embodiments, suppressing an immune response and/or inducing tolerance comprises reducing an allergic response. For example, reducing an allergic response can include reducing an immune response to or inducing tolerance to an antigen associated with allergic asthma, atopic dermatitis, allergic rhinitis (hay fever), or food allergy. In some embodiments, the antigen is an antigen associated with the transplanted tissue. In some embodiments, suppressing an immune response and/or inducing tolerance comprises reducing an immune response or inducing tolerance to the transplanted tissue. In some embodiments, the antigen is associated with a virus. In some embodiments, suppressing an immune response and/or inducing tolerance comprises reducing a pathogenic immune response to a virus or inducing tolerance. For example, a pathogenic immune response can include a cytokine storm produced by certain viral infections. A cytokine storm is a potentially lethal immune response consisting of a positive feedback loop between cytokines and leukocytes.

In some embodiments, suppressing an immune response comprises reducing an immune response to a therapeutic agent. In some embodiments, the therapeutic agent is a clotting factor. Exemplary clotting factors include, but are not limited to, Factor VIII and Factor IX. In some embodiments, a therapeutic agent is an antibody. Exemplary therapeutic antibodies include, but are not limited to, anti-TNFα, anti-VEGF, anti-CD3, anti-CD20, anti-IL-2R, anti-Her2, anti-RSVF, anti-CEA, anti-IL-1 Beta, anti-CD15, anti-myosin, anti-PSMA, anti-40 kDa glycoprotein, anti-CD33, anti-CD52, anti-IgE, anti-CD11a, anti-EGFR, anti-C5, anti-alpha-4 integrins, anti-IL-12/IL-23, anti-IL-6R, and anti-RANKL. In some embodiments, the therapeutic agent is a growth factor. Exemplary therapeutic growth factors include, but are not limited to, erythropoietin (EPO) and megakaryocytic differentiation and growth factor (MDGF). In some embodiments, the therapeutic agent is a hormone. Exemplary therapeutic hormones include, but are not limited to, insulin, human growth hormone, and follicle stimulating hormone. In some embodiments, the therapeutic agent is a recombinant cytokine. Exemplary therapeutic recombinant cytokines include, but are not limited to, IFNβ, IFNα, and GM-CSF. In some embodiments, suppressing the immune response comprises reducing the immune response to the therapeutic vehicle. In some embodiments, the therapeutic vehicle is a virus used in gene therapy, such as an adenovirus, adeno-associated virus, baculovirus, herpes virus, or retrovirus. In some embodiments, the therapeutic vehicle is a lipid-based vehicle, such as a liposome. In some embodiments, the therapeutic vehicle is a nanoparticle.

IV. Microfluidic channels providing cell-modified constrictions

In some embodiments, the invention is a method of suppressing an immune response or inducing tolerance by passing a cell suspension through a constriction, wherein the constriction modifies immune cells, thereby causing perturbation of the cells so that antigens or tolerogenic factors are released into the cells. , wherein the constriction is contained within the microfluidic channel. In some embodiments, multiple constrictions can be placed in parallel and/or series within a microfluidic channel. An exemplary microfluidic channel containing a cell-modified constriction for use in the methods disclosed herein is described in WO2013059343. An exemplary surface with pores for use in the methods disclosed herein is described in US provisional application Ser. No. 62/214,820, filed September 4, 2015.

In some embodiments, the microfluidic channel comprises a lumen and is configured to allow passage of cells suspended in a buffer, wherein the microfluidic channel comprises a constriction. The microfluidic channel may be silicon, metal (eg, stainless steel), plastic (eg, polystyrene), ceramic, glass, crystalline substrate, amorphous substrate, or polymer (eg, poly-methyl methacrylate (PMMA)). ), PDMS, cyclic olefin copolymer (COC), etc.). Fabrication of the microfluidic channel may be performed by any method known in the art including dry etching, wet etching, photolithography, injection molding, laser ablation, or SU-8 masking.

In some embodiments, a constriction within a microfluidic channel includes an inlet portion, a center point, and an outlet portion. In some embodiments, the length, depth, and width of a constriction within a microfluidic channel can vary. In some embodiments, the diameter of a constriction within a microfluidic channel is a function of the diameter of a cell or cell cluster. In some embodiments, the diameter of the constriction within the microfluidic channel is between about 20% and about 99% of the cell diameter. In some embodiments, the constriction size is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter. In some embodiments, the constriction size is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the smallest cross sectional distance of the cells. to be. In some embodiments, the channel comprises a constriction width of from about 2 μm to about 10 μm or any width or range of widths therebetween. For example, the constriction width may be any of about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, or about 7 μm. In some embodiments, the channel comprises a constriction length of about 10 μm and a constriction width of about 4 μm. Cross-sections of the channels, inlet portion, center point, and outlet portion may also vary. For example, the cross-section may be circular, elliptical, elongated, square, hexagonal, or triangular in shape. The inlet portion defines the constriction angle, which is optimized to reduce channel blockage and to enhance delivery of the compound into cells. The angle of the exit portion may also vary. For example, the angle of the exit portion is configured to reduce the possibility of turbulence that can result in non-laminar flow. In some embodiments, the walls of the inlet portion and/or outlet portion are straight. In other embodiments, the walls of the inlet portion and/or outlet portion are curved.

V. Porous surfaces providing cell transformation constrictions

In some embodiments, the invention is a method of suppressing an immune response or inducing tolerance by passing a cell suspension through a constriction, wherein the constriction modifies immune cells, resulting in cell perturbation, resulting in antigenic and/or tolerogenic factors. enters a cell, wherein the constriction is or is contained within a pore. In some embodiments, the pores are contained within the surface. An exemplary surface with pores for use in the methods disclosed herein is described in US provisional application Ser. No. 62/214,820, filed September 4, 2015.

A surface as disclosed herein may be made of any of a number of materials and may take any of a number of forms. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane. In some embodiments, the filter is a tangential flow filter. In some embodiments, the surface is a sponge or sponge-like matrix. In some embodiments, the surface is a matrix.

In some embodiments, the surface is a bend path surface. In some embodiments, the bend path surface comprises cellulose acetate. In some embodiments, the surface is a synthetic or natural polymer, polycarbonate, silicone, glass, metal, alloy, cellulose nitrate, silver, cellulose acetate, nylon, polyester, polyethersulfone, polyacrylonitrile, without limitation. (PAN), polypropylene, PVDF, polytetrafluoroethylene, mixed cellulose esters, porcelain, and ceramics.

A surface disclosed herein may be of any shape known in the art, such as a three-dimensional shape. The two-dimensional shape of the surface can be, without limitation, a circle, ellipse, sphere, rectangle, star, triangle, polygon, pentagon, hexagon, heptagon, or octagon. In some embodiments, the surface is spherical in shape. In some embodiments, the three-dimensional shape of the surface is cylindrical, conical, or cubic.

The surface may vary in cross-sectional width and thickness. In some embodiments, the cross-sectional width of the surface is from about 1 mm to about 1 m, or any cross-sectional width or range of cross-sectional widths therebetween. In some embodiments, the surface has a defined thickness. In some embodiments, the surface thickness is uniform. In some embodiments, the surface thickness is variable. For example, in some embodiments, portions of the surface are thicker or thinner than other portions of the surface. In some embodiments, the surface thickness varies from about 1% to about 90% or any percentage or percentage range in between. In some embodiments, the surface is about 0.01 μm to about 5 mm thick or any thickness or thickness range in between.

In some embodiments, the constriction is a pore or is contained within a pore. The cross-sectional width of the pore is related to the type of cells to be treated. In some embodiments, pore size is a function of the diameter of the cells or cell clusters to be treated. In some embodiments, the pore size is such that cells are disturbed when passing through the pore. In some embodiments, the pore size is less than the cell diameter. In some embodiments, the pore size is between about 10% and about 99% of the cell diameter. In some embodiments, the pore size is about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the cell diameter. , or about 99%. Optimal pore size or pore cross-sectional width may vary based on application and/or cell type. In some embodiments, the pore size is between about 2 μm and about 14 μm. In some embodiments, the pore size is about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 8 μm, about 10 μm, about 12 μm, or about 14 μm. In some embodiments, the cross-sectional width is between about 2 μm and about 14 μm. In some embodiments, the pore cross section is about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 8 μm, about 10 μm, about 12 μm, or about 14 μm.

The inlet and outlet of the pore passage may vary in angle. The pore angle can be selected to minimize pore blockage during cell passage. In some embodiments, the flow rate through the surface is from about 0.001 mL/cm 2 /s to about 100 L/cm 2 /s or any rate or rate range in between. For example, the angle of the inlet or outlet portion may be from about 0 to about 90 degrees. In some embodiments, the inlet or outlet portion may be greater than 90 degrees. In some embodiments, the pores have equal inlet and outlet angles. In some embodiments, the pores have different inlet and outlet angles. In some embodiments, the pore edges are smooth, eg rounded or curved. A smooth pore edge has a continuous, flat and uniform surface without humps, ridges or unevenness. In some embodiments, the pore edges are sharp. Sharp pore edges have pointed or acute thin edges. In some embodiments, the pore passages are straight. Straight pore passages do not contain curves, bends, angles, or other irregularities. In some embodiments, the pore passage is curved. The curved pore passages bend or deviate from straight lines. In some embodiments, the pore passageway has multiple curves, for example about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more curves.

The pores may be of any shape known in the art, including two-dimensional or three-dimensional shapes. The pore shape (eg, cross-sectional shape) can be, without limitation, round, oval, spherical, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal. In some embodiments, the cross section of the pore is spherical in shape. In some embodiments, the three-dimensional shape of the pores is cylindrical or conical. In some embodiments, the pores have a fluted inlet and outlet shape. In some embodiments, the pore shape is homogeneous (ie, consistent or regular) among the pores within a given surface. In some embodiments, the pore shape is not homogeneous (ie, mixed or varied) among pores within a given surface.

The surfaces described herein may vary in total pore count. In some embodiments, the pores comprise from about 10% to about 80% of the total surface area. In some embodiments, the surface contains a total number of pores from about 1.0x10 ⁵ to about 1.0x10 ³⁰ or any number or range of numbers in between. In some embodiments, the surface comprises from about 10 to about 1.0x10 ¹⁵ pores per mm 2 surface area.

The pores can be distributed in a number of ways within a given surface. In some embodiments, the pores are distributed in parallel within a given surface. In one such example, the pores are distributed side by side in the same direction and spaced equidistantly within a given surface. In some embodiments, the pore distribution is orderly or homogeneous. In one such example, the pores are distributed in a regular and organized pattern, or equally spaced apart within a given surface. In some embodiments, the pore distribution is random or non-homogeneous. In one such example, the pores are distributed in an irregular and disordered pattern, or fall at different distances within a given surface. In some embodiments, multiple surfaces are distributed in line. Multiple surfaces can be homogeneous or non-homogeneous in surface size, shape and/or roughness. The multiple surfaces may further contain pores that are homogeneous or heterogeneous in pore size, shape and/or number, thereby allowing simultaneous delivery of a wide range of compounds into different cell types.

In some embodiments, individual pores are uniform in width dimension (ie, constant in width along the length of the pore passageway). In some embodiments, individual pores are variable in width (ie, increase or decrease in width along the length of the pore passageway). In some embodiments, pores within a given surface are equal in individual pore depth. In some embodiments, pores within a given surface differ in individual pore depths. In some embodiments, the pores are adjacent to each other. In some embodiments, the pores are spaced apart from each other. In some embodiments, the pores are separated from each other by a distance of from about 0.001 μm to about 30 mm or any distance or range of distances therebetween.

In some embodiments, the surface is coated with a material. Materials known in the art include, but are not limited to, Teflon, adhesive coatings, surfactants, proteins, adhesion molecules, antibodies, anticoagulants, factors that modulate cellular function, nucleic acids, lipids, carbohydrates, or transmembrane proteins. It can be selected from any material. In some embodiments, the surface is coated with polyvinylpyrrolidone. In some embodiments, the material is covalently attached to the surface. In some embodiments, the material is non-covalently attached to the surface. In some embodiments, surface molecules are released when cells pass through pores.

In some embodiments, the surface has modified chemistry. In some embodiments, the surface is polar. In some embodiments, the surface is hydrophilic. In some embodiments, the surface is non-polar. In some embodiments, the surface is hydrophobic. In some embodiments, the surface is charged. In some embodiments, the surface is positively and/or negatively charged. In some embodiments, the surface may be positively charged in some areas and negatively charged in other areas. In some embodiments, the surface has an overall positive charge or an overall negative charge. In some embodiments, the surface can be any of smooth, electropolished, rough, or plasma treated. In some embodiments, the surface comprises a zwitterionic or dipolar compound. In some embodiments, the surface is plasma treated.

In some embodiments, surfaces are contained within larger modules. In some embodiments, the surface is contained within a syringe, such as a plastic or glass syringe. In some embodiments, the surface is contained within a plastic filter holder. In some embodiments, the surface is contained within a pipette tip.

VI. cell disruption

In some embodiments, the invention is a method of suppressing an immune response or inducing tolerance by passing a cell suspension through a constriction, wherein the constriction modifies immune cells, causing perturbation of the cells so that an antigen or tolerogenic factor is released into the cells. where a disturbance in a cell is a gap (e.g., hole, tear, cavity, interstice, pore, fracture, fissure, perforation) within the cell that allows material from outside the cell to move into the cell. provides a way Deformation can be caused by pressure induced by, for example, mechanical strain and/or shear forces. In some embodiments, a perturbation is a perturbation within a cell membrane. In some embodiments, a perturbation is transient. In some embodiments, the cell perturbation lasts from about 1.0x10 ⁻⁹ seconds to about 2 hours, or any time or time range in between. In some embodiments, the cell perturbation lasts between about 1.0x10 ⁻⁹ seconds and about 1 second, between about 1 second and about 1 minute, or between about 1 minute and about 1 hour. In some embodiments, the cell perturbation is from about 1.0x10 ^-9 sec to about 1.0x10 ^-1 sec, from about 1.0x10 ^-9 sec to about 1.0x10 ^-2 sec, from about 1.0x10 ^-9 sec to about 1.0x10 ^-3 sec, About 1.0x10 ^-9 seconds to about 1.0x10 ^-4 seconds, about 1.0x10 ^-9 seconds to about 1.0x10 ^-5 seconds, about 1.0x10 ^-9 seconds to about 1.0x10 ^-6 seconds, about 1.0x10 ^-9 seconds to about 1.0x10 ^-7 seconds, or any between about 1.0x10 ^-9 seconds to about 1.0x10 ^-8 seconds. In some embodiments, the cell perturbation is from about 1.0x10 ^-8 sec to about 1.0x10 ^-1 sec, from about 1.0x10 ^-7 sec to about 1.0x10 ^-1 sec, from about 1.0x10 ^-6 sec to about 1.0x10 ^-1 sec, From about 1.0x10 ^-5 seconds to about 1.0x10 ^-1 seconds, from about 1.0x10 ^-4 seconds to about 1.0x10 ^-1 seconds, from about 1.0x10 ^-3 seconds to about 1.0x10 ^-1 seconds, or from about 1.0x10 ^-2 seconds Lasts for a random period of about 1.0x10 ^-1 seconds. Cellular disturbances (e.g., pores or pores) produced by the methods described herein do not form as a result of the assembly of protein subunits to form multimeric pore structures such as those produced by complement or bacterial hemolysins. .

As cells pass through the constriction, the constriction temporarily imparts damage to the cell membrane, which causes passive diffusion of substances through perturbation. In some embodiments, the cell is modified only for a period as short as 100 μs to minimize the possibility of activating an apoptotic pathway through a cell signaling mechanism, although other time periods (eg, ranging from nanoseconds to several hours) are possible. . In some embodiments, the cells are deformed for about 1.0x10 ⁻⁹ seconds to about 2 hours, or any time or time range in between. In some embodiments, the cell is deformed for about 1.0x10 ⁻⁹ seconds to about 1 second, about 1 second to about 1 minute, or about 1 minute to about 1 hour. In some embodiments, the cell is about 1.0x10 ^-9 sec to about 1.0x10 ^-1 sec, about 1.0x10 ^-9 sec to about 1.0x10 ^-2 sec, about 1.0x10 ^-9 sec to about 1.0x10 ^-3 sec, about 1.0x10 ^-9 seconds to about 1.0x10 ^-4 seconds, about 1.0x10 ^-9 seconds to about 1.0x10 ^-5 seconds, about 1.0x10 ^-9 seconds to about 1.0x10 ^-6 seconds, about 1.0x10 ^-9 seconds to about 1.0 x10 ^-7 seconds, or any between about 1.0x10 ^-9 seconds to about 1.0x10 ^-8 seconds. In some embodiments, the cell is about 1.0x10 ^-8 sec to about 1.0x10 ^-1 sec, about 1.0x10 ^-7 sec to about 1.0x10 ^-1 sec, about 1.0x10 ^-6 sec to about 1.0x10 ^-1 sec, about 1.0x10 ^-5 seconds to about 1.0x10 ^-1 seconds, about 1.0x10 ^-4 seconds to about 1.0x10 ^-1 seconds, about 1.0x10 ^-3 seconds to about 1.0x10 ^-1 seconds, or about 1.0x10 ^-2 seconds to about Deformed for a random period of 1.0x10 ^-1 sec. In some embodiments, modifying a cell is for a time ranging from, but not limited to, about 1 μs to at least about 750 μs, for example, for at least about 1 μs, 10 μs, 50 μs, 100 μs, 500 μs, or 750 μs. including transforming cells.

In some embodiments, passage of the compound into the cell occurs concurrently with the cell passing through the constriction and/or disrupting the cell. In some embodiments, passage of the compound into the cell occurs after the cell passes through the constriction. In some embodiments, passage of the compound into the cell occurs on the order of minutes after the cell has passed through the constriction. In some embodiments, passage of the compound into the cell occurs between about 1.0x10 ⁻² seconds and at least about 30 minutes after the cell has passed through the constriction. For example, passage of the compound into the cell occurs between about 1.0x10 ^-2 sec and about 1 sec, between about 1 sec and about 1 min, or between about 1 min and about 30 min after the cell has passed through the constriction. In some embodiments, passage of the compound into the cell is between about 1.0x10 ^-2 seconds and about 10 minutes, between about 1.0x10 ^-2 seconds and about 5 minutes, between about 1.0x10 ^-2 seconds and about 1 minute after the cells pass through the constriction. ^{. _ _ _} In some embodiments, passage of the compound into the cell is from about 1.0x10 ⁻¹ sec to about 10 min, from about 1 sec to about 10 min, from about 10 sec to about 10 min, from about 50 sec to about 10 min after the cell has passed the constriction. 10 minutes, about 1 minute to about 10 minutes, or about 5 minutes to about 10 minutes. In some embodiments, a disturbance in a cell after the cell has passed the constriction is corrected within about 5 minutes or less after the cell has passed the constriction.

In some embodiments, cell viability after crossing the constriction is between about 5% and about 100%. In some embodiments, cell viability after crossing the constriction is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% , 95%, or 99%. In some embodiments, cell viability is measured from about 1.0x10 ⁻² seconds to at least about 10 days after cells pass through the constriction. For example, cell viability can be determined from about 1.0x10 ^-2 seconds to about 1 second, from about 1 second to about 1 minute, from about 1 minute to about 30 minutes, or from about 30 minutes to about 2 hours after the cells pass through the constriction. It is measured. In some embodiments, the cell viability is between about 1.0x10 ^-2 seconds and about 2 hours, between about 1.0x10 ^-2 seconds and about 1 hour, between about 1.0x10 ^-2 seconds and about 30 minutes, about 1.0 x10 ⁻² seconds to about 1 minute, about 1.0×10 ⁻² seconds to about 30 seconds, about 1.0×10 ⁻² seconds to about 1 second, or about 1.0×10 ⁻² seconds to about 0.1 seconds. In some embodiments, the cell viability is from about 1.5 hours to about 2 hours, from about 1 hour to about 2 hours, from about 30 minutes to about 2 hours, from about 15 minutes to about 2 hours, from about 1 minute after the cells pass the constriction. to about 2 hours, about 30 seconds to about 2 hours, or about 1 second to about 2 hours. In some embodiments, cell viability is measured between about 2 hours and about 5 hours, between about 5 hours and about 12 hours, between about 12 hours and about 24 hours, or between about 24 hours and about 10 days after the cells pass through the constriction. .

VII. transfer parameter

A number of parameters can affect the delivery of a compound to a cell to suppress an immune response or induce tolerance by the methods described herein. In some embodiments, the cell suspension is contacted with the compound before, concurrently with, or after passage through the stenosis. Although the compound may be added to the cell suspension after the cells have passed through the constriction, the cells may pass through the constriction suspended in a solution containing the compound to be delivered. In some embodiments, the compound to be delivered is coated onto the stricture.

Examples of parameters that can affect the delivery of a compound into cells include the dimensions of the constriction, the angle of entrance to the constriction, the surface properties of the constriction (eg, roughness, chemical modification, hydrophilicity, hydrophobicity, etc.), working flow rate (eg, , constriction transit time), cell concentration, concentration of the compound in the cell suspension, including but not limited to, the amount of time the cells are withdrawn or incubated after passing through the constriction, the amount of time the delivered compound is dependent on passage into the cell. can affect Additional parameters that affect delivery of the compound into the cells may include the velocity of the cells within the constriction, the shear rate within the constriction, the viscosity of the cell suspension, the velocity component perpendicular to the flow rate, and the time in the constriction. These parameters can be designed to control compound delivery. In some embodiments, the cell concentration ranges from about 10 to at least about 10 ¹² cells/ml or any concentration or concentration range in between. In some embodiments, the delivery compound concentration may range from about 10 ng/ml to about 1 g/mL or any concentration or concentration range in between. In some embodiments, the delivery compound concentration may range from about 1 pM to at least about 2 M or any concentration or concentration range in between.

Temperatures used in the methods of the present disclosure can be adjusted to affect compound delivery and cell viability. In some embodiments, the method is performed at about -5°C to about 45°C. For example, the method may be room temperature (e.g., about 20°C), physiological temperature (e.g., about 37°C), a temperature above physiological temperature (e.g., greater than about 37°C to 45°C or above), or at a lower temperature (eg, from about -5°C to about 4°C), or at a temperature between these exemplary temperatures.

A variety of methods are available for driving cells through the constriction. For example, pressure may be applied by a pump (eg, gas cylinder, or compressor) on the inlet side, vacuum may be applied by a vacuum pump on the outlet side, and/or capillary tubes may be applied through tubing. Actions can be applied and/or gravity can be supplied to the system. Displacement based flow systems can also be used (eg, syringe pumps, peristaltic pumps, hand syringes or pipettes, pistons, etc.). In some embodiments, the cells pass through the constriction with positive or negative pressure. In some embodiments, the cells pass through the constriction with constant or variable pressure. In some embodiments, pressure is applied using a syringe. In some embodiments, pressure is applied using a pump. In some embodiments, the pump is a peristaltic pump or pump or diaphragm pump. In some embodiments, pressure is applied using a vacuum. In some embodiments, the cell passes through the constriction with a g-force. In some embodiments, the cells pass through the constriction by centrifugal force. In some embodiments, the cells pass through the constriction by capillary pressure.

In some embodiments, fluid flow directs the cells through the constriction. In some embodiments, the fluid flow is turbulent before the cells pass through the constriction. Turbulence is a fluid flow in which the velocity at a given point varies unsteadily in magnitude and direction. In some embodiments, fluid flow through the constriction is laminar. Laminar flow involves uninterrupted flow in a fluid near a solid boundary where the direction of flow at all points remains constant. In some embodiments, the fluid flow is turbulent after the cells pass through the constriction. The rate at which cells pass through the constriction can vary. In some embodiments, the cells pass through the constriction at a uniform cell velocity. In some embodiments, the cells pass through the constriction at a fluctuating cell speed.

In another embodiment, the cell suspension is passed through the constriction, and the constriction modifies immune cells, causing perturbation of the cells, thereby suppressing the immune response or inducing tolerance by allowing antigens or tolerogenic factors to enter the cells. used, for example, followed by exposure to an electric field downstream of the constriction in the method described herein. In some embodiments, the cell is passed through an electric field generated by at least one electrode after passing through the constriction. In some embodiments, the electric field assists in delivering the compound to a second location inside the cell, such as the cell nucleus. For example, the combination of a cell-modifying constriction and an electric field delivers a plasmid encoding the antibody into a cell (eg, cell nucleus), resulting in de novo production of the antibody. In some embodiments, one or more electrodes are adjacent to the cell-modified constriction to generate an electric field. In some embodiments, the electric field is from about 0.1 kV/m to about 100 MV/m, or any number or range of numbers therebetween. In some embodiments, an integrated circuit is used to provide electrical signals to drive the electrodes. In some embodiments, the cell is exposed to the electric field with a pulse width of about 1 ns to about 1 s and a duration of about 100 ns to about 10 s or any time or time range in between.

VIII. Cell suspension for delivery to immune cells

A cell suspension can be a mixed or purified cell population. In some embodiments, the cell suspension is a mixed cell population, such as whole blood. In some embodiments, the cell suspension is a purified cell population, such as a purified immune cell population.

The composition of the cell suspension (eg, osmolarity, salt concentration, serum content, cell concentration, pH, etc.) can affect the delivery of a compound to suppress an immune response or induce tolerance. In some embodiments, the suspension comprises whole blood. Alternatively, a cell suspension is a mixture of cells in a physiological medium other than saline solution or blood. In some embodiments, the cell suspension comprises an aqueous solution. In some embodiments, the aqueous solution is added to cell culture medium, PBS, salts, sugars, growth factors, animal derived products, bulking materials, surfactants, lubricants, vitamins, amino acids, proteins, cell cycle inhibitors, and/or actin polymerization. Including agents that affect In some embodiments, the cell culture medium is DMEM, Opti-MEM®, IMDM, or RPMI. Additionally, the solution buffer may include one or more lubricants (pluronics or other surfactants), which may be designed, for example, to reduce or eliminate surface clogging and improve cell viability. Exemplary surfactants include, but are not limited to, poloxamers, polysorbates, sugars or sugar alcohols such as mannitol, sorbitol, animal derived serum, and albumin proteins.

In some configurations with certain cell types, the cells may be incubated in one or more solutions that aid in the delivery of the compound into the cells. In some embodiments, the aqueous solution includes an agent that affects actin polymerization. In some embodiments, the agent that affects actin polymerization is latrunculin A, cytochalasin, and/or colchicine. For example, cells can be incubated in a depolymerization solution such as latrunculin A (0.1 μg/ml) for 1 hour prior to transfer to depolymerize the actin cytoskeleton. As a further example, cells can be incubated in 10 μM Colchicine (Sigma) for 2 hours prior to transfer to depolymerize the microtubule network.

In some embodiments, a cell population is enriched prior to use in a disclosed method. For example, cells are obtained from a bodily fluid, such as peripheral blood, and optionally enriched or purified to enrich for B cells. Cells can be enriched by any method known in the art, including but not limited to magnetic cell separation, fluorescence activated cell sorting (FACS), or density gradient centrifugation.

Viscosity of the cell suspension can also affect the methods disclosed herein. In some embodiments, the viscosity of the cell suspension ranges from about 8.9x10 ^-4 Pa.s to about 4.0x10 ^-3 Pa.s or any value or range of values therebetween. In some embodiments, the viscosity ranges from about 8.9x10 ^-4 Pa.s to about 4.0 x10 ^-3 Pa.s, from about 8.9x10 ^-4 Pa.s to about 3.0 x10 ^-3 Pa.s, from about 8.9x10 ^-4 Pa.s. s to about 2.0 x10 ^-3 Pa.s, or from about 8.9 x10 -3 Pa.s to about 1.0 x10 ^-3 Pa.s. In some embodiments, the viscosity range is between any of about 0.89 cP to about 4.0 cP, about 0.89 cP to about 3.0 cP, about 0.89 cP to about 2.0 cP, or about 0.89 cP to about 1.0 cP. In some embodiments, a shear thinning effect is observed where the viscosity of the cell suspension is reduced under conditions of shear deformation. Viscosity can be measured by any method known in the art, including but not limited to a viscometer, such as a glass capillary viscometer, or a galvanometer. Viscometers measure viscosity under one flow condition, while galvanometers are used to measure viscosity that changes with flow conditions. In some embodiments, viscosity is measured for shear thinning solutions such as blood. In some embodiments, the viscosity is measured between about -5°C and about 45°C. For example, room temperature (e.g., about 20°C), physiological temperature (e.g., about 37°C), temperature above physiological temperature (e.g., greater than about 37°C to 45°C or greater) , a low temperature (eg, about -5° C. to about 4° C.), or a temperature between these exemplary temperatures.

IX. Antigens and tolerogenic factors for suppressing the immune response or inducing tolerance

In some embodiments, the invention provides delivery of an antigen to suppress an immune response or induce tolerance, wherein the antigen is delivered to a cell by any of the methods described herein. In some embodiments, an antigen is a single antigen. In some embodiments, the antigen is a mixture of antigens. An antigen is a substance that stimulates a specific immune response, such as a cell or antibody-mediated immune response. Antigens bind to receptors expressed by immune cells, such as the T cell receptor (TCR), which are specific for a particular antigen. Antigen-receptor binding then triggers intracellular signaling pathways that lead to downstream immune effector pathways such as cell activation, cytokine production, cell migration, cytotoxic factor secretion, and antibody production.

In some embodiments, the antigen is a polypeptide antigen. In some embodiments, the compound comprises a disease-associated antigen. In some embodiments, the antigen is from a foreign source, such as a bacterium, fungus, virus, or allergen. In some embodiments, the antigen is derived from an internal source, such as a self-protein (ie, self-antigen). In some embodiments, the antigen is in a cell lysate. Self-antigens are antigens present on or within the cells of an organism's own. Self-antigens do not normally stimulate an immune response, but may in the context of autoimmune diseases such as type I diabetes or rheumatoid arthritis. In some embodiments, an antigen is a therapeutic agent. In some embodiments, an antigen is a therapeutic polypeptide or fragment of a therapeutic polypeptide. In some embodiments, the therapeutic agent is a clotting factor. Exemplary clotting factors include, but are not limited to, Factor VIII and Factor IX. For example, the antigen may be a factor VIII protein used in the treatment of hemophilia. In some embodiments, a therapeutic agent is an antibody. Exemplary therapeutic antibodies include, but are not limited to, anti-TNFα, anti-VEGF, anti-CD3, anti-CD20, anti-IL-2R, anti-Her2, anti-RSVF, anti-CEA, anti-IL-1 Beta, anti-CD15, anti-myosin, anti-PSMA, anti-40 kDa glycoprotein, anti-CD33, anti-CD52, anti-IgE, anti-CD11a, anti-EGFR, anti-C5, anti-alpha-4 integrins, anti-IL-12/IL-23, anti-IL-6R, and anti-RANKL. In some embodiments, the therapeutic agent is a growth factor. Exemplary therapeutic growth factors include, but are not limited to, erythropoietin (EPO) and megakaryocytic differentiation and growth factor (MDGF). In some embodiments, the therapeutic agent is a hormone. Exemplary therapeutic hormones include, but are not limited to, insulin, human growth hormone, and follicle stimulating hormone. In some embodiments, the therapeutic agent is a recombinant cytokine. Exemplary therapeutic recombinant cytokines include, but are not limited to, IFNβ, IFNα, and GM-CSF.

In some embodiments, the antigen is an allograft transplant antigen. Allograft transplantation is the transfer of cells, tissues or organs from a genetically non-identical donor of the same species to a recipient. In some embodiments, the antigen is a modified antigen. For example, an antigen can be fused with a therapeutic agent or targeting peptide. In some embodiments, a modified antigen is fused to a polypeptide. In some embodiments, the modified antigen is fused with a lipid. In some embodiments, the antigen is a non-protein antigen, such as a lipid, glycolipid, or polysaccharide. In some embodiments, the antigen is a whole microorganism, such as an intact bacterium.

In some embodiments, the antigen is associated with a virus. In some embodiments, the antigen is a viral antigen. Exemplary viral antigens include SARS-CoV antigens and influenza antigens. In some embodiments, the compound comprises a cell lysate from tissue infected with an unknown pathogen. In some embodiments, the antigen is a therapeutic vehicle. In some embodiments, the therapeutic vehicle is a virus used in gene therapy, such as an adenovirus, adeno-associated virus, baculovirus, herpes virus, or retrovirus. In some embodiments, the therapeutic vehicle is a liposome. In some embodiments, the therapeutic vehicle is a nanoparticle.

In some embodiments, the antigen is associated with a microorganism, such as a bacterium. In some embodiments, suppressing an immune response and/or inducing tolerance comprises reducing or inducing a pathogenic immune response against a microorganism, eg, a bacterium. In some embodiments, a microorganism is part of an individual's microbiome. In some embodiments, a microorganism may be beneficial to an individual's microbiome.

In certain aspects, the invention is a method of delivering an antigen into immune cells, comprising passing a cell suspension comprising immune cells through a constriction, wherein the constriction modifies the immune cells, thereby causing disruption of the cells so that the antigen is transferred to the cells. wherein the cell suspension is contacted with an antigen. In some embodiments, processing and presentation of an antigen in a tolerogenic environment suppresses an immune response to the antigen. In some embodiments, processing and presentation of the antigen in a tolerogenic environment induces tolerance to the antigen. In some embodiments, antigens are delivered to immune cells in vitro, ex vivo, or in vivo.

In some embodiments, the present invention provides delivery of tolerogenic factors to suppress an immune response or induce tolerance, wherein the tolerogenic factors are delivered to cells by any of the methods described herein. In some embodiments, the tolerogenic factor enhances the suppression of an immune response to the antigen or enhances the induction of tolerance to the antigen. For example, tolerogenic factors can promote tolerogenic presentation of antigens by antigen-presenting cells. In some embodiments, the tolerogenic factor is introduced simultaneously with the antigen. In some embodiments, the tolerogenic factor and antigen are introduced sequentially.

In certain aspects, the invention is a method of generating immunosuppressive antigen-presenting immune cells comprising an antigen, wherein the immunosuppressive immune cells pass through a constriction, wherein the constriction causes disruption of the cells by transforming them, wherein the antigen enters an immune cell, thereby generating immunosuppressive immune cells comprising the antigen.

In some embodiments, the tolerogenic factor modulates the expression and/or activity of an immunomodulatory agent (such as an immunostimulatory agent (eg, a co-stimulatory molecule), an immunosuppressive agent, or an inflammatory or anti-inflammatory molecule). In some embodiments, the tolerogenic factor inhibits the expression and/or activity of an immunostimulatory agent (eg, a co-stimulatory molecule), enhances the expression and/or activity of an immunosuppressive molecule, and/or inflammatory molecule inhibits the expression and/or activity of, and/or enhances the expression and/or activity of anti-inflammatory molecules. In some embodiments, the tolerogenic factor inhibits the activity of a costimulatory molecule. Interactions between costimulatory molecules and their ligands are important for sustaining and integrating TCR signaling to stimulate optimal T cell proliferation and differentiation. In some embodiments, the tolerogenic factor reduces expression of costimulatory molecules. Exemplary costimulatory molecules expressed on antigen-presenting cells include, but are not limited to, CD40, CD80, CD86, CD54, CD83, CD79, or ICOS ligands. In some embodiments, the costimulatory molecule is CD80 or CD86. In some embodiments, the tolerogenic factor inhibits the expression of a nucleic acid that expresses or modulates the expression of a costimulatory molecule. In some embodiments, the tolerogenic factor deletes a nucleic acid that expresses or modulates the expression of a costimulatory molecule. In some embodiments, deletion of a nucleic acid that expresses or modulates the expression of a costimulatory molecule is achieved through gene editing. In some embodiments, the tolerogenic factor inhibits a costimulatory molecule. In some embodiments, the tolerogenic factor is a siRNA that inhibits a costimulatory molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that inhibits the expression of costimulatory molecules. In some embodiments, the tolerogenic factor increases the activity of a protein inhibitor that inhibits expression of costimulatory molecules. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an inhibitor of a costimulatory molecule. In some embodiments, the tolerogenic factor degrades the costimulatory molecule. In some embodiments, the tolerogenic factor labels costimulatory molecules for destruction. For example, tolerogenic factors can enhance the ubiquitination of costimulatory molecules, thereby targeting them for destruction.

In some embodiments, the tolerogenic factor enhances the expression and/or activity of an immunosuppressive molecule. In some embodiments, the immunosuppressive molecule is a co-inhibitory molecule, transcriptional regulator, or immunosuppressive molecule. Co-inhibitory molecules negatively regulate lymphocyte activation. Exemplary co-inhibitory molecules include, but are not limited to, PD-L1, PD-L2, HVEM, B7-H3, TRAIL, immunoglobulin-like transcript (ILT) receptors (ILT2, ILT3, ILT4), FasL, CTLA4, CD39, CD73, and B7-H4. In some embodiments, the co-inhibitory molecule is PD-L1 or PD-L2. In some embodiments, the tolerogenic factor increases the activity of the co-inhibitory molecule. In some embodiments, the tolerogenic factor increases expression of co-inhibitory molecules. In some embodiments, the tolerogenic factor encodes a co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the activity of the co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that enhances the expression of co-inhibitory molecules. In some embodiments, the tolerogenic factor increases the activity of the polypeptide to increase expression of co-inhibitory molecules. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an enhancer of a co-inhibitory molecule. In some embodiments, the tolerogenic factor inhibits the inhibitor of the co-inhibitory molecule.

In some embodiments, the tolerogenic factor increases the expression and/or activity of an immunosuppressive molecule. Exemplary immunosuppressive molecules include, but are not limited to, arginase-1 (ARG1), indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), Nitric oxide (NO), nitric oxide synthase 2 (NOS2), thymic stromal lymphopoietin (TSLP), vascular intestinal peptide (VIP), hepatocyte growth factor (HGF), transforming growth factor beta (TGFβ), IFNα, IL-4, IL-10, IL-13, and IL-35. In some embodiments, the immunosuppressive molecule is NO or IDO. In some embodiments, the tolerogenic factor encodes an immunosuppressive molecule. In some embodiments, the tolerogenic factor increases the activity of an immunosuppressive molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that enhances the expression of immunosuppressive molecules. In some embodiments, the tolerogenic factor increases the activity of the polypeptide to enhance expression of immunosuppressive molecules. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an enhancer of an immunosuppressive molecule. In some embodiments, the tolerogenic factor inhibits a negative regulator of an immunosuppressive molecule.

In some embodiments, the tolerogenic factor inhibits the expression and/or activity of an inflammatory molecule. In some embodiments, the inflammatory molecule is an inflammatory transcription factor. In some embodiments, the tolerogenic factor inhibits an inflammatory transcription factor. In some embodiments, the tolerogenic factor reduces expression of an inflammatory transcription factor. In some embodiments, the inflammatory transcription factor is a molecule associated with NF-κB, interferon regulatory factor (IRF), or JAK-STAT signaling pathway. The NF-κB pathway is a pro-inflammatory signaling pathway that mediates the expression of pro-inflammatory genes including cytokines, chemokines and adhesion molecules. Interferon regulatory factors (IRFs) constitute a family of transcription factors that can regulate the expression of pro-inflammatory genes. The JAK-STAT signaling pathway transmits information from extracellular cytokine signals to the nucleus, resulting in DNA transcription and expression of genes involved in immune cell proliferation and differentiation. The JAK-STAT system consists of a cell surface receptor, Janus kinase (JAK), and signal transducer and activator of transcription (STAT) proteins. Exemplary JAK-STAT molecules include, but are not limited to, JAK1, JAK2, JAK 3, Tyk2, STAT1, STAT2, STAT3, STAT4, STAT5 (STAT5A and STAT5B), and STAT6. In some embodiments, the tolerogenic factor enhances expression of an inhibitor of cytokine signaling (SOCS) protein. SOCS proteins can inhibit signaling through the JAK-STAT pathway. In some embodiments, the tolerogenic factor inhibits expression of a nucleic acid encoding an inflammatory transcription factor. In some embodiments, the tolerogenic factor deletes a nucleic acid encoding an inflammatory transcription factor. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that inhibits the expression of an inflammatory transcription factor. In some embodiments, the tolerogenic factor increases the activity of a protein inhibitor that inhibits the expression of an inflammatory transcription factor. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an inhibitor of an inflammatory transcription factor.

In some embodiments, the tolerogenic factor enhances the expression and/or activity of anti-inflammatory molecules. In some embodiments, the anti-inflammatory molecule is an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor enhances an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor increases expression of an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor enhances expression of a nucleic acid encoding an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor reduces the activity of a transcriptional regulator that inhibits expression of an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor reduces the activity of a protein inhibitor that inhibits expression of an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an enhancer of an anti-inflammatory transcription factor.

In some embodiments, the tolerogenic factor comprises a nucleic acid. In some embodiments, the tolerogenic factor is a nucleic acid. Exemplary nucleic acids include, but are not limited to, recombinant nucleic acids, DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, saRNA, miRNA, lncRNA, tRNA, guide RNA, and shRNA. In some embodiments, a nucleic acid is homologous to a nucleic acid in a cell. In some embodiments, the nucleic acid is heterologous to the nucleic acid in the cell. In some embodiments, the tolerogenic factor is a plasmid. In some embodiments, the nucleic acid is a therapeutic nucleic acid. In some embodiments, the nucleic acid encodes a therapeutic polypeptide. In some embodiments, tolerogenic factors include nucleic acids encoding siRNA, mRNA, miRNA, lncRNA, tRNA, or shRNA. For example, tolerogenic factors can include siRNAs that knock-down the expression of inflammatory genes. In some embodiments, the tolerogenic factor is a DNA sequence that binds to NFκB and prevents NFκB activation and downstream signaling.

In some embodiments, the tolerogenic factor comprises a polypeptide. In some embodiments, the tolerogenic factor is a polypeptide. In some embodiments, the protein or polypeptide is a therapeutic protein, antibody, fusion protein, antigen, synthetic protein, reporter marker, or selectable marker. In some embodiments, the protein is a gene-editing protein or nuclease such as zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, CRE recombinase, trans propellase, CAS9 enzyme, or integrase enzyme. In some embodiments, the fusion protein may include, but is not limited to, a chimeric protein drug such as an antibody drug conjugate or a recombinant fusion protein such as a GST or streptavidin tagged protein. In some embodiments, the compound is a transcription factor. Exemplary transcription factors include, but are not limited to, Oct5, Sox2, c-Myc, Klf-4, T-bet, GATA3, FoxP3, and RORγt. In some embodiments, the polypeptide is IL-4, IL-10, IL-13, IL-35, IFNα, or TGFβ. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the polypeptide is a fragment of a therapeutic polypeptide. In some embodiments, a polypeptide is a peptide nucleic acid (PNA).

In some embodiments, the tolerogenic factor comprises a protein-nucleic acid complex. In some embodiments, the tolerogenic factor is a protein-nucleic acid complex. In some embodiments, protein-nucleic acid complexes such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 are used for genome editing applications. These complexes contain sequence-specific DNA-binding domains in combination with non-specific DNA cleavage nucleases. These complexes enable targeted genome editing, including adding, destroying or changing the sequence of specific genes. In some embodiments, impaired CRISPR is used to block or induce transcription of a target gene. In some embodiments, the tolerogenic factor contains a Cas9 protein and guide RNA or donor DNA. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding a Cas9 protein and a guide RNA or donor DNA. In some embodiments, the gene editing complex targets expression of costimulatory molecules (eg, CD80 and/or CD86).

In some embodiments, the tolerogenic factor comprises a Chimeric Antigen Receptor (CAR). In some embodiments, the tolerogenic factor is a Chimeric Antigen Receptor (CAR). In some embodiments, a CAR is a fusion of an extracellular recognition domain (eg, an antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains. Upon engagement with an antigen, the intracellular signaling portion of the CAR can initiate an immunosuppressive or tolerogenic-related response in immune cells. In some embodiments, the CAR is a chimeric T cell antigen receptor. In some embodiments, the CAR antigen-binding domain is a single chain antibody variable fragment (scFv). In some embodiments, the tolerogenic factor encodes a modified TCR containing a cytoplasmic signaling domain that upon binding to antigen triggers the production of immunosuppressive cytokines. In some embodiments, the tolerogenic factor encodes a chimeric antigen receptor that contains a cytoplasmic signaling domain that upon binding to the antigen triggers the production of an immunosuppressive cytokine.

In some embodiments, tolerogenic factors include small molecules. In some embodiments, the tolerogenic factor is a small molecule. In some embodiments, the small molecule inhibits the activity of a costimulatory molecule, enhances the activity of a co-inhibitory molecule, and/or inhibits the activity of an inflammatory molecule. Exemplary small molecules include, but are not limited to, pharmaceuticals, metabolites, and radionuclides or radionuclide-containing molecules. In some embodiments, the medicament is a therapeutic drug and/or cytotoxic agent. In some embodiments, the compound comprises nanoparticles. Examples of nanoparticles include gold nanoparticles, quantum dots, carbon nanotubes, nanoshells, dendrimers, and liposomes. In some embodiments, nanoparticles contain or are linked (covalently or non-covalently) to therapeutic molecules. In some embodiments, nanoparticles contain nucleic acids, such as mRNA or cDNA.

In certain aspects, the invention is a method of delivering tolerogenic factors into immune cells, comprising passing a cell suspension comprising immune cells through a constriction, wherein the constriction modifies the immune cells, thereby causing disruption of the cells to allow tolerance. The tolerogenic factor enters the cell, wherein the cell suspension is contacted with the tolerogenic factor. In some embodiments, tolerogenic factors are delivered to immune cells in vitro, ex vivo, or in vivo.

In some embodiments, the compound to be delivered is purified. In some embodiments, the compound is at least about 60% by weight (dry weight) of the compound of interest. In some embodiments, the purified compound is at least about 75%, 90%, or 99% of the compound of interest. In some embodiments, the purified compound is at least about 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the compound of interest. Purity is determined by any known method including but not limited to column chromatography, thin layer chromatography, HPLC analysis, NMR, mass spectrometry, or SDS-PAGE. Purified DNA or RNA is defined as DNA or RNA free of exogenous nucleic acids, carbohydrates and lipids.

In some embodiments, the compound is an intermediate compound. An intermediate compound may be a molecular entity that is formed from a preceding intermediate and reacts to further provide a final reaction product. In some embodiments, an intermediate compound is a protein precursor, or whole protein, which is enzymatically cleaved to produce a mature functional form of the protein. In some embodiments, an intermediate compound is an inactive enzyme precursor, or enzyme source, which requires modification or cleavage to produce an active enzyme.

X. Use

In some aspects, the invention provides a method of treating a patient by introducing modified immune cells into the patient by passing the compound through a constriction to allow entry into the cells. In some embodiments, treatment comprises introducing such modified immune cells into the patient multiple times (such as any of 2, 3, 4, 5, 6, or more). In some embodiments, cells are isolated from a patient, modified according to the disclosed methods, and introduced back into the patient. For example, an immune cell population is isolated from a patient, passed through a stricture to achieve compound delivery, and then reintroduced into the patient to augment a therapeutic immune response. In some embodiments, cells are isolated from an individual, modified according to the disclosed methods, and reintroduced into the individual. For example, an immune cell population is isolated from a subject, passed through a stricture to achieve compound delivery, and then reintroduced into a patient to suppress an immune response or induce tolerance in the subject.

In some embodiments, the invention provides a method of treating an individual by introducing modified cells into the individual by passing the compound through a constriction to allow entry into the cells. In some embodiments, the cells are autologous cells. For example, immune cells are isolated from a subject (eg, a patient), modified according to the disclosed methods, and reintroduced into the subject. In some embodiments, cells are isolated from an individual, modified according to the disclosed methods, and reintroduced into the same individual. In some embodiments, the cell is an allogeneic cell. For example, cells are isolated from a different subject, modified according to the disclosed methods, and introduced into a first subject (eg, patient). In some embodiments, pools of cells from multiple individuals are modified according to the disclosed methods and introduced into a first subject (eg, patient). In some embodiments, cells are isolated from an individual, modified according to the disclosed methods, and introduced into a different individual. In some embodiments, a population of cells is isolated from an individual (patient) or a different individual, passed through the stricture to achieve delivery of a compound that induces production of di-novo antibodies, and then injected into the patient to enhance the therapeutic response.

In some embodiments, treatment comprises multiple (eg, any of 2, 3, 4, 5, 6, or more) administrations of modified immune cells as described herein to the individual. For example, in some embodiments, a method of treating an individual is provided by administering to the individual two, three, four, five, six or more times cells modified by passing the compound through the constriction to enter the cells. do. In some embodiments, the period between any two consecutive cell administrations is at least about 1 day (such as at least about 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 months, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or more (including any range between these values) ) is any of them).

Any of the methods described above are performed in vitro, ex vivo, or in vivo. For in vivo use, the device can be implanted into the lumen of a blood vessel, for example an in-line stent can be implanted into an artery or vein. In some embodiments, the method is used as part of a bedside system for ex vivo treatment of patient cells and immediate reintroduction of cells into a patient. Such methods can be used as a means to suppress an immune response or induce tolerance in a subject. In some embodiments, the methods can be performed by minimally trained technicians in a typical hospital laboratory. In some embodiments, a patient manipulation treatment system may be used. In some embodiments, the method is practiced using an in-line blood therapy system in which blood is bypassed directly from the patient, passed through the stenosis, resulting in delivery of the compound to blood cells, and transfused directly back into the patient after treatment. do.

XI. systems and kits

In some aspects, the invention provides a system comprising a constriction, a cell suspension and a compound for use in a method disclosed herein. Such systems may be any of the embodiments described for the methods disclosed above, including microfluidic channels or pore surfaces that provide cell-modified constrictions, cell suspensions, cell perturbations, delivery parameters, compounds, and/or uses, and the like. can include In some embodiments, the cell-modified constriction is sized for delivery to immune cells. In some embodiments, delivery parameters such as working flow rate, cell and compound concentration, rate of cells at the constriction, and composition of the cell suspension (e.g., , osmolarity, salt concentration, serum content, cell concentration, pH, etc.) are optimized.

Kits or articles of manufacture for use in delivering compounds to suppress an immune response or induce tolerance are also provided. In some embodiments, a kit includes a composition described herein (eg, a surface containing microfluidic channels or pores, a cell suspension, and/or a compound) in suitable packaging. Suitable packaging materials are known in the art and include, for example, vials (eg, sealed vials), containers, ampoules, bottles, jars, flexible packaging (eg, sealed mylar or plastic bags), and the like. Such articles of manufacture may further be sterilized and/or sealed.

The invention may also provide kits comprising components of the methods described herein, and may further include instructions(s) for performing the methods to suppress an immune response or induce tolerance. Kits described herein may be packaged with other buffers, diluents, filters, needles, syringes, and instructions for performing any of the methods described herein, eg, suppressing an immune response or inducing tolerance. It may further include other materials, including inserts.

XII. Exemplary Embodiments

Embodiment 1. A method of inducing tolerance to an antigen in an individual,

a. passing a cell suspension comprising immune cells through a constriction, wherein the constriction deforms the cells, causing disturbance of the cells so that antigens contacted with the cells enter the immune cells; and

b. Introducing immune cells into a subject

Including,

wherein the immune cells are tolerogenic immune cells, wherein the presentation of the antigen induces tolerance to the antigen.

Way.

Embodiment 2. A method of inducing tolerance to an antigen in an individual,

a. A cell suspension comprising immune cells is passed through a constriction, wherein the constriction transforms the cells, thereby causing disturbance of the cells so that antigens and tolerogenic factors contacted with the cells enter the immune cells, whereby the tolerogenicity including the antigens is achieved. generating immune cells; and

b. Introducing tolerogenic immune cells into a subject

Including,

wherein the presentation of the antigen induces tolerance to the antigen.

Way.

Embodiment 3. A method of suppressing an immune response to an antigen in an individual,

a. passing a cell suspension comprising immune cells through a constriction, wherein the constriction deforms the cells, causing disturbance of the cells so that antigens contacted with the cells enter the immune cells; and

b. Introducing immune cells into a subject

Including,

wherein the immune cells are tolerogenic immune cells, wherein the presentation of the antigen suppresses an immune response to the antigen.

Way.

Embodiment 4. A method of suppressing an immune response to an antigen in an individual,

a. A cell suspension comprising immune cells is passed through a constriction, wherein the constriction transforms the cells, causing disturbance of the cells so that antigens and tolerogenic factors contacted with the cells enter the immune cells, whereby immunosuppression including the antigens is performed. generating sexual immune cells; and

b. Introducing immunosuppressive immune cells into a subject

Including,

Wherein the presentation of the antigen is to suppress the immune response to the antigen.

Way.

Embodiment 5. A method of inducing tolerance to an antigen in an individual comprising introducing tolerogenic immune cells comprising the antigen into the individual, wherein presentation of the antigen induces tolerance to the antigen, wherein the antigen is The method wherein the tolerogenic immune cells or precursors thereof were introduced into tolerogenic immune cells by passing them through the constriction, wherein the constriction transforms the cells to cause disturbance of the cells so that the antigen enters the tolerogenic immune cells or precursors thereof. .

Embodiment 6. A method of inducing tolerance to an antigen in an individual, wherein tolerogenic immune cells comprising the antigen and tolerogenic factors are introduced into the individual, wherein the presentation of the antigen induces tolerance to the antigen, wherein the antigen and tolerogenic factors were introduced into the tolerogenic immune cells by passing the tolerogenic immune cells or their precursors to the constriction, wherein the constriction transforms the cells, causing disturbance of the cells so that the antigens and tolerogenic factors are introduced into the tolerogenic immune cells. Or a method comprising entering a precursor thereof.

Embodiment 7. A method of suppressing an immune response to an antigen in an individual, wherein immunosuppressive immune cells comprising the antigen are introduced into the individual, wherein the presentation of the antigen suppresses an immune response to the antigen, wherein the antigen is Immunosuppressive immune cells or their precursors were introduced into the immunosuppressive immune cells by passing them through the constriction, wherein the constriction caused cell disruption by transforming the cells so that antigens entered the immunosuppressive immune cells or their precursors. A method comprising steps.

Embodiment 8. A method of suppressing an immune response to an antigen in a subject, wherein immunosuppressive immune cells comprising the antigen and tolerogenic factors are introduced into the subject, wherein the presentation of the antigen suppresses the immune response to the antigen; wherein the antigen and tolerogenic factor are introduced into the immunosuppressive immune cell by passing the immunosuppressive immune cell or a precursor thereof into the constriction, wherein the constriction transforms the cell to cause cell disruption so that the antigen and tolerogenic factor are immunosuppressive A method comprising entering a sexual immune cell or a precursor thereof.

Embodiment 9. A method of producing tolerogenic immune cells, comprising passing a cell suspension comprising immune cells through a constriction, wherein the constriction causes disturbance of the cells by modifying the cells, thereby causing tolerogenic factors contacted with the cells. wherein enters immune cells, thereby generating tolerogenic immune cells.

Embodiment 10. A method of generating immunosuppressive immune cells, comprising passing a cell suspension comprising immune cells through a constriction, wherein the constriction modifies the cells, thereby causing perturbation of the cells, resulting in tolerogenic properties in contact with the cells. wherein the factor enters the immune cell, thereby generating immunosuppressive immune cells.

Embodiment 11. The method according to embodiment 9 or 10, wherein the constriction transforms the cells, causing disturbance of the cells so that tolerogenic factors and antigens contacted with the cells enter immune cells.

Embodiment 12. The method of embodiment 9 or 10, wherein the immune cell further passes through a second constriction, wherein the second constriction transforms the cell, causing disturbance of the cell, such that an antigen contacted with the cell enters the immune cell. how it would be.

Embodiment 13. The method of any one of embodiments 9-12, wherein the immune cells are antigen-presenting cells.

Embodiment 14. A method of generating tolerogenic antigen-presenting cells comprising an antigen, wherein the tolerogenic antigen-presenting cells pass through a constriction, wherein the constriction transforms the cells, thereby causing disturbance of the cells so that they come into contact with the cells. wherein the antigen enters an antigen-presenting cell.

Embodiment 15. A method of generating an immunosuppressive antigen-presenting cell comprising an antigen, wherein the immunosuppressive antigen-presenting cell passes through a constriction, wherein the constriction causes disruption of the cell by modifying the cell, thereby wherein the contacted antigen enters an antigen-presenting cell.

Embodiment 16. A method of delivering a tolerogenic factor that produces a tolerogenic phenotype into immune cells, wherein a cell suspension comprising immune cells is passed through a constriction, wherein the constriction modifies the immune cells, thereby causing perturbation of the cells, resulting in tolerance wherein the tolerogenic factor enters the cell, and contacting the cell suspension with a tolerogenic factor, wherein the tolerogenic factor inhibits the expression and/or activity of an immunostimulatory agent and/or an immunosuppressive molecule. The method of enhancing the expression and/or activity of, inhibiting the expression and/or activity of an inflammatory molecule, and/or enhancing the expression and/or activity of an anti-inflammatory molecule.

Embodiment 17. A method of delivering an antigen into tolerogenic immune cells, wherein a cell suspension comprising tolerogenic immune cells is passed through a constriction, wherein the constriction modifies the tolerogenic immune cells, thereby causing disruption of the cells so that the antigen is transferred to the cells and contacting the cell suspension with an antigen.

Embodiment 18. A method of delivering a tolerogenic factor that produces an immunosuppressive phenotype into immune cells, wherein a cell suspension comprising immune cells is passed through a constriction, wherein the constriction modifies the immune cells, causing disruption of the cells, wherein the tolerogenic factor enters the cell, and contacting the cell suspension with the tolerogenic factor, wherein the tolerogenic factor inhibits the expression and/or activity of an immunostimulatory agent and/or has an immunosuppressive effect. enhancing the expression and/or activity of the molecule, inhibiting the expression and/or activity of the inflammatory molecule, and/or enhancing the expression and/or activity of the anti-inflammatory molecule.

Embodiment 19. A method of delivering an antigen into immunosuppressive immune cells, wherein a cell suspension comprising immunosuppressive immune cells is passed through a constriction, wherein the constriction modifies the immunosuppressive immune cells, thereby causing disruption of the cells A method comprising the step of allowing the antigen to enter the cell, and contacting the cell suspension with the antigen.

Embodiment 20. A method of suppressing an immune response to an antigen in an individual,

a. passing the first cell suspension comprising the first immune cells through the constriction, wherein the constriction transforms the cells to cause disturbance of the cells so that antigens contacted with the cells enter the immune cells;

b. A second cell suspension comprising a second immune cell is passed through the constriction, wherein the constriction deforms the cells, thereby causing disturbance of the cells so that tolerogenic factors contacted with the cells enter the immune cells, thereby producing immunosuppressive immunity. generating cells; and

c. introducing a first immune cell and a second immune cell into a subject;

Including,

Wherein the presentation of the antigen is to suppress the immune response to the antigen.

Way.

Embodiment 21. The method of embodiment 20, wherein the presentation of said antigen by said first or second immune cell suppresses an immune response to the antigen.

Embodiment 22. The method of embodiment 20, wherein the second immune cell imparts an immunosuppressive phenotype on the first immune cell to generate an immunosuppressive first immune cell, and wherein the immunosuppressive first immune cell of the antigen wherein the presentation by suppresses an immune response to the antigen.

Embodiment 23. A method of inducing tolerance to an antigen in an individual,

a. passing the first cell suspension comprising the first immune cells through the constriction, wherein the constriction transforms the cells to cause disturbance of the cells so that antigens contacted with the cells enter the immune cells;

b. A second cell suspension comprising a second immune cell is passed through the constriction, wherein the constriction causes disturbance of the cells by deforming the cells so that tolerogenic factors contacted with the cells enter the immune cells, whereby the tolerogenic immune cells The step of generating; and

c. introducing a first immune cell and a second immune cell into a subject;

Including,

Wherein the presentation of the antigen is to suppress the immune response to the antigen.

Way.

Embodiment 24. The method of embodiment 23, wherein presentation of said antigen by said first or second immune cell induces tolerance to the antigen.

Embodiment 25. The method of embodiment 23, wherein the second immune cell confer an tolerogenic phenotype on the first immune cell to produce an tolerogenic first immune cell, and the presentation of said antigen by said tolerogenic first immune cell. A method of inducing tolerance to the antigen.

Embodiment 26. The method of any one of embodiments 20-25, wherein the first immune cell and the second immune cell are introduced into the subject simultaneously.

Embodiment 27. The method of any one of embodiments 20-25, wherein the first immune cell and the second immune cell are introduced into the subject sequentially.

Embodiment 28. according to any one of embodiments 1, 2, 5, 6, 9, 11-14, 16, 17, and 23-27, wherein the tolerogenic immune cell or precursor thereof is not contacted with an adjuvant. Way.

Embodiment 29. according to any one of embodiments 3, 4, 7, 8, 10-13, 15, 18-22, 26, and 27, wherein the immunosuppressive immune cell or precursor thereof is not contacted with an adjuvant way of being.

Embodiment 30. The method according to embodiment 28 or 29, wherein the adjuvant is a TLR3 and RLR ligand, a TLR4 ligand, a TLR5 ligand, a TLR7/8 ligand, a TLR9 ligand, a NOD2 ligand, alum, a water-in-oil emulsion, rhIL-2, an anti-CD40 , CD40L, IL-12, and a method selected from the group consisting of cyclic dinucleotides.

Embodiment 31. The method of any one of embodiments 1, 2, 5, 6, 9, 11-14, 16, 17, and 23-28, wherein the tolerogenic immune cell is directed to a non-tolerogenic precursor of the tolerogenic immune cell. and a reduced ability to provide one or more costimulatory signals when compared.

Embodiment 32. The immunosuppressive immune cell according to any one of embodiments 3, 4, 7, 8, 10-13, 15, 18-22, 26, 27, and 29, wherein the immunosuppressive immune cell is non- and a reduced ability to provide one or more costimulatory signals as compared to an immunosuppressive precursor.

Embodiment 33. The method of embodiment 31 or 32, wherein the one or more costimulatory signals are mediated by a molecule selected from the group consisting of CD40, CD80, CD86, CD54, CD83, CD79, and ICOS ligands.

Embodiment 34. The tolerogenic immune cell according to any one of embodiments 1, 2, 5, 6, 9, 11-14, 16, 17, 23-28, and 31, wherein the tolerogenic immune cell has a non-tolerogenic effect A method comprising a reduced ability to provide one or more inflammatory signals as compared to a precursor.

Embodiment 35. The immunosuppressive immune cell according to any one of embodiments 3, 4, 7, 8, 10-13, 15, 18-22, 26, 27, 29, and 32, wherein the immunosuppressive immune cell is and a reduced ability to provide one or more inflammatory signals as compared to a non-immunosuppressive precursor.

Embodiment 36. The method according to embodiment 34 or 35, wherein the one or more inflammatory signals are interleukin-1 (IL-1), IL-12, IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma), granulocytes -mediated by a molecule selected from the group consisting of macrophage colony stimulating factor (GM-CSF), NF-κB, interferon regulatory factor (IRF), and molecules associated with the JAK-STAT signaling pathway.

Embodiment 37. The method according to any one of embodiments 2, 4, 6, 8-11, 16, 18, 20, and 23, wherein the tolerogenic factor inhibits the expression and/or activity of an immunostimulant and/or is immunosuppressive. enhancing the expression and/or activity of inflammatory molecules, inhibiting the expression and/or activity of inflammatory molecules, and/or enhancing the expression and/or activity of anti-inflammatory molecules.

Embodiment 38. The method of any one of embodiments 1, 2, 5, 6, 11-14, 17, 23-28, and 31, wherein the antigen is presented by tolerogenic immune cells.

Embodiment 39. according to any one of embodiments 3, 4, 7, 8, 11-13, 15, 19-22, 26, 27, 29, and 32, wherein the antigen is presented by an immunosuppressive immune cell way of being.

Embodiment 40. The method of any one of embodiments 2, 6, 9, 11-13, 16, 23-28, and 31, wherein tolerance to at least one other antigen is induced.

Embodiment 41. The method of any one of embodiments 4, 8, 10-13, 18, 20-22, 26, 27, 29, and 32, wherein an immune response to at least one other antigen is suppressed.

Embodiment 42. The method of any one of embodiments 1-8, 11, 12, 14, 15, 17, 19, and 20-25, wherein at least one additional antigen is introduced into the cell.

Embodiment 43. The method of any one of embodiments 2, 4, 6, 8, 9-11, 16, 18, 20, and 23, wherein at least one additional tolerogenic factor is introduced into the cell.

Embodiment 44. A method of suppressing an immune response in an individual,

a. passing the cell suspension comprising immune cells through a constriction, wherein the constriction deforms the cells, causing disturbance of the cells so that a compound encoding a non-functional cytokine binding protein contacted with the cells enters the immune cells; and

b. Introducing immune cells into a subject

Including,

wherein said non-functional cytokine binding protein is expressed, wherein said non-functional cytokine binding protein binds a free inflammatory cytokine.

Way.

Embodiment 45. The method of embodiment 44, wherein the non-functional cytokine binding protein comprises a non-functional cytokine receptor.

Embodiment 46. The method of embodiment 45, wherein the non-functional cytokine receptor lacks a cytoplasmic signaling domain.

Embodiment 47. The method of embodiment 44, wherein the non-functional cytokine binding protein comprises a proteolytic site that cleaves a target cytokine.

Embodiment 48. The method of embodiment 44, wherein the non-functional cytokine binding protein comprises an anti-cytokine antibody.

Embodiment 49. The method of embodiment 44, wherein the non-functional cytokine binding protein comprises an anti-cytokine B cell receptor.

Embodiment 50. The method of any one of embodiments 1-7 and 20-44, wherein the immune cells are from an individual.

Embodiment 51. The method of any one of embodiments 1-7 and 20-44, wherein the immune cells are from different individuals.

Embodiment 52. The method of any one of embodiments 1-51, wherein the constriction is contained within the microfluidic channel.

Embodiment 53. The method of any one of embodiments 1-51, wherein the constriction is or is contained within a pore.

Embodiment 54. The method of embodiment 53, wherein the pores are contained within the surface.

Embodiment 55. The method of embodiment 54, wherein the surface is a filter.

Embodiment 56. The method of embodiment 54, wherein the surface is a membrane.

Embodiment 57. The method of any one of embodiments 1-56, wherein the constriction size is a function of the diameter of an immune cell.

Embodiment 58. The method of any one of embodiments 1-57, wherein the constriction size is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99%.

Embodiment 59. The method of any one of embodiments 1-58, wherein the channel comprises a constriction length of about 10 μm and a constriction width of about 4 μm.

Embodiment 60. The method of any one of embodiments 1-59, wherein the pore size is about 0.4 μm, about 3 μm, about 4 μm, about 5 μm, about 8 μm, about 10 μm, about 12 μm, or about 14 μm Way.

Embodiment 61. The method of any one of embodiments 1-60, wherein the method is performed at about -5°C to about 45°C.

Embodiment 62. The method of any one of embodiments 1-4, 9, 10, and 16-61, wherein the cell suspension comprises a mixed cell population.

Embodiment 63. The method of embodiment 62, wherein the cell suspension is whole blood.

Embodiment 64. The method of embodiment 62, wherein the cell suspension comprises peripheral blood mononuclear cells.

Embodiment 65. The method of any one of embodiments 1-4, 9, 10, and 16-61, wherein the cell suspension comprises a purified cell population.

Embodiment 66. The method of any one of embodiments 1-4, 9, 10, and 16-65, wherein the cell suspension comprises mammalian cells.

Embodiment 67. The method of any one of embodiments 1-4, 9, 10, and 16-66, wherein the cell suspension comprises monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cells. How to do.

Embodiment 68. The method of any one of embodiments 1-4, 9, 10, and 16-66, wherein the cell suspension comprises human cells.

Embodiment 69. The method of any one of embodiments 1-4, 9, 10, and 16-65, wherein the cell suspension comprises non-mammalian cells.

Embodiment 70. The method of any one of embodiments 1-4, 9, 10, 16-65, and 69, wherein the cell suspension comprises bacterial, yeast, chicken, frog, insect, fish, or nematode cells. .

Embodiment 71. The method of any one of embodiments 1-13 and 16-68, wherein the immune cells are mammalian cells.

Embodiment 72. The method of any one of embodiments 1-13, 16-68, and 71, wherein the immune cells are monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cells.

Embodiment 73. The method of any one of embodiments 1-13, 16-68, and 71, wherein the immune cells are human cells.

Embodiment 74. The method according to any one of embodiments 1-13 and 16-73, wherein the immune cell is a T cell, B cell, dendritic cell, monocyte, macrophage, NK cell, innate lymphoid cell, neutrophil, basophil, eosinophil, Bone marrow derived suppressor cells, or mast cells.

Embodiment 75. The method of any one of embodiments 14, 15, and 50-74, wherein the antigen-presenting cell is a mammalian cell.

Embodiment 76. The method of any one of embodiments 14, 15, and 50-75, wherein the antigen-presenting cells are monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit cells.

Embodiment 77. The method of any one of embodiments 14, 15, and 50-75, wherein the antigen-presenting cell is a human cell.

Embodiment 78. The method according to any one of embodiments 14, 15, and 50-77, wherein the antigen-presenting cell is a T cell, B cell, dendritic cell, monocyte, macrophage, NK cell, innate lymphoid cell, neutrophil, basophil , eosinophils, bone marrow derived suppressor cells, or mast cells.

Embodiment 79. The method of any one of embodiments 1-15, 20-43, and 50-78, wherein the antigen is a foreign antigen.

Embodiment 80. The method of any one of embodiments 1-15, 20-43, and 50-78, wherein the antigen is a self-antigen.

Embodiment 81. The method of any one of embodiments 1-15, 20-43, and 50-78, wherein the antigen is an allograft transplant antigen.

Embodiment 82. The method of any one of embodiments 1-15, 20-43, and 50-78, wherein the antigen is a protein or polypeptide.

Embodiment 83. The method of any one of embodiments 1-15, 20-43, and 50-78, wherein the antigen is a lysate.

Embodiment 84. The method of any one of embodiments 1-15, 20-43, and 50-83, wherein the antigen is a modified antigen.

Embodiment 85. The method of embodiment 84, wherein the modified antigen comprises an antigen fused with a therapeutic agent.

Embodiment 86. The method of embodiment 84, wherein the modified antigen comprises an antigen fused with a targeting peptide.

Embodiment 87. The method of any one of embodiments 1-4, 9, 10, and 16-86, wherein said cell suspension is contacted with the antigen before, simultaneously with, or after passage through the stenosis.

Embodiment 88. The method of any one of embodiments 1-43 and 50-87, wherein the tolerogenic factor inhibits the activity of a costimulatory molecule.

Embodiment 89. The method of any one of embodiments 1-43 and 50-88, wherein the tolerogenic factor reduces expression of a costimulatory molecule.

Embodiment 90. The method of any one of embodiments 1-43 and 50-89, wherein the tolerogenic factor deletes a nucleic acid that modulates expression of a costimulatory molecule.

Embodiment 91. The method of any one of embodiments 1-43 and 50-88, wherein the tolerogenic factor inhibits a costimulatory molecule.

Embodiment 92. The method of any one of embodiments 1-43 and 50-91, wherein the tolerogenic factor increases the activity of a transcriptional regulator that inhibits the expression of costimulatory molecules.

Embodiment 93. The method of any one of embodiments 1-43 and 50-91, wherein the tolerogenic factor increases the activity of a protein inhibitor that inhibits expression of a costimulatory molecule.

Embodiment 94. The method of any one of embodiments 1-43 and 50-88, wherein the tolerogenic factor comprises a nucleic acid encoding an inhibitor of a costimulatory molecule.

Embodiment 95. The method of any one of embodiments 88-94, wherein the costimulatory molecule is CD80 or CD86.

Embodiment 96. The method of any one of embodiments 1-43 and 50-95, wherein the tolerogenic factor potentiates the activity of an immunosuppressive factor.

Embodiment 97. The method of embodiment 96, wherein the immunosuppressive factor is a co-inhibitory molecule, a transcriptional regulator, or an immunosuppressive molecule.

Embodiment 98. The method of any one of embodiments 1-43, 50-87, 96, and 97, wherein the tolerogenic factor potentiates the activity of a co-inhibitory molecule.

Embodiment 99. The method of any one of embodiments 1-43, 50-87, and 96-98, wherein the tolerogenic factor increases expression of a co-inhibitory molecule.

Embodiment 100. The method of any one of embodiments 1-43, 50-87, and 96-99, wherein the tolerogenic factor encodes a co-inhibitory molecule.

Embodiment 101. The method of any one of embodiments 1-43, 50-87, and 96-100, wherein the tolerogenic factor increases the activity of the co-inhibitory molecule.

Embodiment 102. The method of any one of embodiments 1-43, 50-87, and 96-98, wherein the tolerogenic factor increases the activity of a transcriptional regulator that enhances expression of a co-inhibitory molecule.

Embodiment 103. The method of any one of embodiments 1-43, 50-87, 96-98, and 102, wherein the tolerogenic factor increases the activity of a polypeptide that increases expression of a co-inhibitory molecule.

Embodiment 104. The method of any one of embodiments 1-43, 50-87, 96-98, and 102, wherein the tolerogenic factor comprises a nucleic acid encoding an enhancer of a co-inhibitory molecule.

Embodiment 105. The method of any one of embodiments 97-104, wherein the co-inhibitory molecule is PD-L1, PD-L2, or CTLA-4.

Embodiment 106. The method of any one of embodiments 1-43, 50-87, 96, and 97, wherein the tolerogenic factor enhances the activity of an immunosuppressive molecule.

Embodiment 107. The method of any one of embodiments 1-43, 50-87, 96, 97, and 106, wherein the tolerogenic factor increases expression of an immunosuppressive molecule.

Embodiment 108. The method of any one of embodiments 1-43, 50-87, 96, 97, and 106, wherein the tolerogenic factor encodes an immunosuppressive molecule.

Embodiment 109. The method of any one of embodiments 1-43, 50-87, 96, 97, and 106, wherein the tolerogenic factor increases the activity of the immunosuppressive molecule.

Embodiment 110. The method according to any one of embodiments 1-43, 50-87, 96, 97, and 106, wherein the tolerogenic factor increases the activity of a transcriptional regulator that enhances the expression of an immunosuppressive molecule. .

Embodiment 111. The method of any one of embodiments 1-43, 50-87, 96, 97, and 106, wherein the tolerogenic factor increases the activity of a polypeptide that enhances the expression of an immunosuppressive molecule.

Embodiment 112. The method of any one of embodiments 1-43, 50-87, 96, 97, and 106, wherein the tolerogenic factor comprises a nucleic acid encoding an enhancer of an immunosuppressive molecule.

Embodiment 113. The method of any one of embodiments 97 and 106, wherein the immunosuppressive molecule is ARG1, NO, NOS2, IDO, IL-4, IL-10, IL-13, IL-35, IFNα, or TGFβ .

Embodiment 114. The method of any one of embodiments 1-43 and 50-87, wherein the tolerogenic factor inhibits the activity of an inflammatory molecule.

Embodiment 115. The method of embodiment 114, wherein the inflammatory molecule is an inflammatory transcription factor.

Embodiment 116. The method of any one of embodiments 1-43, 50-87, 114, and 115, wherein the tolerogenic factor inhibits an inflammatory transcription factor.

Embodiment 117. The method of any one of embodiments 1-43, 50-87, and 114-116, wherein the tolerogenic factor reduces expression of an inflammatory transcription factor.

Embodiment 118. The method of any one of embodiments 1-43, 50-87, and 114-116, wherein the tolerogenic factor deletes a nucleic acid encoding an inflammatory transcription factor.

Embodiment 119. The method of any one of embodiments 1-43, 50-87, and 114-116, wherein the tolerogenic factor increases the activity of a transcriptional regulator that inhibits the expression of an inflammatory transcription factor.

Embodiment 120. The method of any one of embodiments 1-43, 50-87, and 114-116, wherein the tolerogenic factor increases the activity of a protein inhibitor that inhibits the expression of an inflammatory transcription factor.

Embodiment 121. The method of any one of embodiments 1-43, 50-87, and 114-116, wherein the tolerogenic factor comprises a nucleic acid encoding an inhibitor of an inflammatory transcription factor.

Embodiment 122. The method of any one of embodiments 115-121, wherein the inflammatory transcription factor is NF-κB, an interferon regulator, or a molecule associated with the JAK-STAT signaling pathway.

Embodiment 123. The method of any one of embodiments 1-43, 50-87, and 114-116, wherein the tolerogenic factor reduces production and/or secretion of one or more inflammatory cytokines.

Embodiment 124. The method according to embodiment 123, wherein the one or more inflammatory cytokines are interleukin-1 (IL-1), IL-12, and IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma), and granulocytes - a method selected from the group consisting of macrophage colony stimulating factor (GM-CSF).

Embodiment 125. The method of any one of embodiments 1-43, 50-87, and 114-116, wherein the tolerogenic factor increases production and/or secretion of one or more anti-inflammatory cytokines.

Embodiment 126. is according to embodiment 125, wherein the one or more anti-inflammatory cytokines are selected from the group consisting of IL-4, IL-10, IL-13, IL-35, IFN-α and transforming growth factor-beta (TGFβ) How to be.

Embodiment 127. is according to any one of embodiments 1-43 and 50-87, wherein the tolerogenic factor encodes a modified TCR containing a cytoplasmic signaling domain that triggers the production of immunosuppressive cytokines upon binding to the antigen. way of being.

Embodiment 128. The method according to any one of embodiments 1-43 and 50-87, wherein the tolerogenic factor encodes a chimeric antigen receptor containing a cytoplasmic signaling domain that triggers production of an immunosuppressive cytokine upon binding to the antigen. way of being.

Embodiment 129. The method of any one of embodiments 1-43 and 50-128, wherein the tolerogenic factor comprises a nucleic acid.

Embodiment 130. The method of any one of embodiments 1-43 and 50-129, wherein the tolerogenic factor comprises a nucleic acid encoding siRNA, mRNA, miRNA, lncRNA, tRNA, or shRNA.

Embodiment 131. The method of any one of embodiments 1-43 and 50-130, wherein the tolerogenic factor is a plasmid.

Embodiment 132. The method of any one of embodiments 1-43 and 50-128, wherein the tolerogenic factor comprises a protein-nucleic acid complex.

Embodiment 133. The method of any one of embodiments 1-43, 50-128, and 132, wherein the tolerogenic factor comprises a Cas9 polypeptide and a guide RNA or donor DNA.

Embodiment 134. The method according to any one of embodiments 1-43 and 50-129, wherein the tolerogenic factor comprises a nucleic acid encoding a Cas9 polypeptide and a guide RNA or donor DNA.

Embodiment 135. The method of any one of embodiments 1-43 and 50-128, wherein the tolerogenic factor comprises a polypeptide.

Embodiment 136. The method of any one of embodiments 1-43, 50-128, and 135, wherein the polypeptide is a nuclease, TALEN protein, zinc finger nuclease, meganuclease, or CRE recombinase.

Embodiment 137. The method of any one of embodiments 1-43, 50-128, and 135, wherein the polypeptide is a transposase or integrase enzyme.

Embodiment 138. The method of any one of embodiments 1-43, 50-128, and 135, wherein the polypeptide is an antibody.

Embodiment 139. The method of any one of embodiments 1-43, 50-128, and 135, wherein the polypeptide is a transcription factor.

Embodiment 140. The method of any one of embodiments 1-43 and 50-128, wherein the tolerogenic factor is a small molecule.

Embodiment 141. The method according to any one of embodiments 1-43 and 50-128, wherein the tolerogenic factor is a nanoparticle.

Embodiment 142. The method according to any one of embodiments 1-43 and 50-141, wherein said cell suspension is contacted with tolerogenic factors before, concurrently with, or after passage through the stenosis.

Embodiment 143. The method of any one of embodiments 3, 4, 7, 8, and 10-142, wherein the immune response is at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40% %, about 50%, about 60%, about 70%, about 75%, about 80%, about 90%, or about 100%.

Embodiment 144. The method of any one of embodiments 3, 4, 7, 8, and 10-143, wherein suppressing the immune response comprises reducing production and/or secretion of one or more inflammatory cytokines.

Embodiment 145. The method according to embodiment 144, wherein the one or more inflammatory cytokines are interleukin-1 (IL-1), IL-12, and IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma), and granulocyte-macrophage colony stimulating factor (GM-CSF).

Embodiment 146. The method according to any one of embodiments 3, 4, 7, 8, and 10-143, wherein suppressing the immune response comprises increasing production and/or secretion of one or more anti-inflammatory cytokines.

Embodiment 147. is according to embodiment 146, wherein the one or more anti-inflammatory cytokines are selected from the group consisting of IL-4, IL-10, IL-13, IL-35, IFN-α and transforming growth factor-beta (TGFβ) How to be.

Embodiment 148. The method of any one of embodiments 3, 4, 7, 8, and 10-143, wherein suppressing the immune response comprises reducing a T cell response.

Embodiment 149. The method of embodiment 148, wherein reducing a T cell response comprises reducing T cell activation.

Embodiment 150. The method of embodiment 148 or 149, wherein reducing the T cell response comprises reducing T cell survival.

Embodiment 151. The method of any one of embodiments 148-150, wherein reducing the T cell response comprises reducing T cell proliferation.

Embodiment 152. The method of any one of embodiments 148-151, wherein reducing the T cell response comprises reducing T cell functionality.

Embodiment 153. The method of any one of embodiments 3, 4, 7, 8, and 10-152, wherein suppressing the immune response comprises enhancing a Treg response.

Embodiment 154. The method of any one of embodiments 3, 4, 7, 8, and 10-153, wherein suppressing the immune response comprises reducing a B cell response.

Embodiment 155. The method of embodiment 154, wherein reducing the B cell response comprises reducing antibody production.

Embodiment 156. The method of any one of embodiments 3, 4, 7, 8, and 10-155, wherein suppressing the immune response comprises reducing cytokine production.

Embodiment 157. The method of any one of embodiments 3, 4, 7, 8, and 10-156, wherein suppressing the immune response comprises reducing an autoimmune response.

Embodiment 158. The method of any one of embodiments 3, 4, 7, 8, and 10-157, wherein suppressing the immune response comprises reducing an allergic response.

Embodiment 159. The method of any one of embodiments 1-15, 20-43, and 50-78, wherein the antigen is an antigen associated with a transplanted tissue.

Embodiment 160. The method of any one of embodiments 3, 4, 7, 8, and 10-159, wherein suppressing the immune response comprises reducing the immune response to the transplanted tissue.

Embodiment 161. The method of any one of embodiments 1-15, 20-43, and 50-78, wherein the antigen is associated with a virus.

Embodiment 162. The method of any one of embodiments 3, 4, 7, 8, and 10-161, wherein suppressing the immune response comprises reducing a pathogenic immune response to the virus.

Embodiment 163. The method of any one of embodiments 3, 4, 7, 8, and 10-162, wherein suppressing the immune response comprises reducing the immune response to the therapeutic agent.

Embodiment 164. The method of any one of embodiments 3, 4, 7, 8, and 10-163, wherein suppressing the immune response comprises reducing the immune response to the therapeutic vehicle.

Embodiment 165. The method of any one of embodiments 1, 2, 5, 6, 23-43, and 50-142, wherein tolerance comprises reducing production and/or secretion of one or more inflammatory cytokines.

Embodiment 166. The method according to embodiment 165, wherein the one or more inflammatory cytokines are interleukin-1 (IL-1), IL-12, and IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma), and granulocytes - a method selected from the group consisting of macrophage colony stimulating factor (GM-CSF).

Embodiment 167. The method of any one of embodiments 1, 2, 5, 6, 23-43, and 50-142, wherein tolerance comprises increased production and/or secretion of one or more anti-inflammatory cytokines.

Embodiment 168. is according to embodiment 167, wherein the one or more anti-inflammatory cytokines are selected from the group consisting of IL-4, IL-10, IL-13, IL-35, IFN-α and transforming growth factor-beta (TGFβ) How to be.

Embodiment 169. The method of any one of embodiments 1, 2, 5, 6, 23-43, and 50-142, wherein the tolerance comprises reducing a T cell response.

Embodiment 170. The method of embodiment 169, wherein reducing a T cell response comprises reducing T cell activation.

Embodiment 171. The method of embodiment 169 or 170, wherein reducing the T cell response comprises reducing T cell survival.

Embodiment 172. The method of any one of embodiments 169-171, wherein reducing the T cell response comprises reducing T cell proliferation.

Embodiment 173. The method of any one of embodiments 169-172, wherein reducing the T cell response comprises reducing T cell functionality.

Embodiment 174. The method of any one of embodiments 169-173, wherein the reduction in T cell response comprises a change in T cell phenotype.

Embodiment 175. The method of any one of embodiments 1, 2, 5, 6, 23-43, 50-142, and 169-174, wherein the tolerance comprises non-costimulatory activation of T cells.

Embodiment 176. The method of any one of embodiments 1, 2, 5, 6, 23-43, 50-142, and 169-175, wherein the tolerance comprises enhancing a Treg response.

Embodiment 177. The method of any one of embodiments 1, 2, 5, 6, 23-43, 50-142, and 169-176, wherein the tolerance comprises reducing a B cell response.

Embodiment 178. The method of embodiment 177, wherein reducing the B cell response comprises reducing antibody production.

Embodiment 179. The method of any one of embodiments 1, 2, 5, 6, 23-43, 50-142, and 169-178, wherein the tolerance comprises reducing cytokine production.

Embodiment 180. The method of any one of embodiments 1, 2, 5, 6, 23-43, 50-142, and 169-179, wherein the tolerance comprises reducing an autoimmune response.

Embodiment 181. The method of any one of embodiments 1, 2, 5, 6, 23-43, 50-142, and 169-180, wherein the tolerance comprises reducing an allergic response.

Embodiment 182. The method of any one of embodiments 1, 2, 5, 6, 23-43, 50-142, and 169-181, wherein the tolerance comprises reducing an immune response to the transplanted tissue.

Embodiment 183. The method of any one of embodiments 1, 2, 5, 6, 23-43, 50-142, and 169-182, wherein the tolerance comprises reducing a pathogenic immune response to the virus.

Embodiment 184. The method of any one of embodiments 1, 2, 5, 6, 23-43, 50-142, and 169-183, wherein the tolerance comprises a reduced immune response to the therapeutic agent.

Embodiment 185. The method of any one of embodiments 1, 2, 5, 6, 23-43, 50-142, and 169-184, wherein the tolerance comprises a reduced immune response to the therapeutic vehicle.

Embodiment 186. The method of any one of embodiments 1-185, wherein the method is repeated at least 1, 2, 3, 4, 5, or 6 times.

Embodiment 187 The method of embodiment 186, wherein the period between any two repetitions of the method is at least 1 day, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or 1 year.

Embodiment 188. A system comprising a stricture, immune cell, antigen, and/or tolerogenic factor for use in the method of any one of embodiments 1-43 and 50-187.

Embodiment 189. A system comprising a constriction, an immune cell, and a compound encoding a non-functional cytokine binding protein for use in the method of any one of embodiments 44-187.

Example

The following examples are provided for the purpose of illustrating various embodiments of the disclosure and are not intended to limit the disclosure in any way. Those skilled in the relevant art will readily appreciate that the present disclosure is well adapted to carry out the objectives and obtain the objects and advantages mentioned, as well as the objects, objects and advantages inherent herein. Variations and other uses herein will occur to those skilled in the art that come within the spirit of the disclosure as defined by the scope of the claims.

Example 1: Constriction-mediated delivery of antigens and tolerogenic factors to B cells to induce tolerance

A series of experiments are undertaken to demonstrate tolerance induction using model antigens and transgenic T cells.

B cells are mixed with tolerogenic factors and OVA antigen +/- tolerogenic factors and pass through constrictions in microfluidic channels, or surfaces containing pores (this constriction-mediated delivery is also referred to as SQZ). Optimize pressure, temperature, and buffer composition to achieve delivery. CFSE-labeled OT-I or OT-II T cells are transferred into CD45.1 mice followed by OVA and tolerogenic factor-loaded B cells. Later, the mice received i.d. of OVA+LPS. Challenged by injection. Draining popliteal lymph nodes are harvested to measure the expansion of OT-I or OT-II T cells.

Example 2: Constriction-mediated delivery of antigens to B cells to induce tolerance

In this study, the ability of B cell antigen-presenting cells (APCs), which contain intracellular antigens generated by constriction-mediated delivery of antigens, to induce antigen-dependent tolerance in vivo was evaluated. Mice were inoculated with antigen-specific T cells, tolerized by injection of B cell APC, and the number of antigen-specific T cells and the level of the inflammatory cytokine IFN-γ were determined.

materials and methods

OVA-specific CD8 ⁺ OT-I cells (1x10 ⁶ ) and CD4 ⁺ OT-II T cells (1x10 ⁶ ) (both CD45.2+) per mouse from female donor mice on day 0 were C57BL/6J Adoptive transfer into recipient mice (CD45.1+). On day 1, B cells from C57BL/6J mice were harvested and incubated with complete OVA protein without constriction (B cell-Endo) or OVA protein was delivered constriction-mediated intracellularly (B cell-SQZ). . Then, 5x10 ⁶ of each B cell/mouse were injected into recipient mice. Control animals were injected with PBS (challenge control) or 10 μg free OVA (tolerance control). Antigen was challenged on day 8 with intradermal injection of 10 μg OVA protein + 50 ng LPS/mouse. Draining lymph nodes and spleens of each mouse were analyzed on day 12, and OT-I and OT-II T cell responses to antigen challenge were analyzed by flow cytometry for the number of OT-I and OT-II T cells, IFN-γ Intracellular staining, and measured by ELI spot assay. A schematic diagram of the treatment schedule is presented in FIG. 1 and the treatment groups are summarized in Table 1.

Table 1

result

OT-I- and OT-II-specific T cell numbers were measured on day 12 in mice from the four treatment groups. Cells from draining lymph nodes (CD3 ⁺ /CD8 ⁺ gating or CD3 ⁺ /CD4 ⁺ gating) and spleen (CD3 ⁺ /CD8 ⁺ gating) were tested for T-cell receptor (TCR) V alpha 2 ( TCRVa2) and staining for CD45.2 as a marker for donor T cells. Activation of OT-I T cells in both draining lymph nodes and spleen, as indicated by a decrease in the percentage of CD8+ OT-I T cells (TCRVa2+/CD45.2+) (Figure 2, left and middle panels) and Proliferation was significantly inhibited in mice primed with B cells with OVA protein stenosis-mediated transfer when compared to challenge controls (* P < 0.05). As shown by reduced levels of CD8+ OT-I T cells in B cell-SQZ animals compared to mice primed with B cells incubated with OVA without strictures (** P < 0.01), this effect is not due to stricture-mediated cell It was dependent on delivery of my antigens. The percentage of CD4+ OT-II cells (TCRVa2+/CD45.2+) was also significantly inhibited in lymph nodes for B cell-SQZ animals when compared to challenge controls (FIG. 2, right panel; * P <0.05). B cell-SQZ animals also showed a slight decrease in the percentage of OT-II after challenge when compared to B cell-Endo with a trend toward significance, while significant differences in splenic OT-II levels between treatment groups were absent (data not shown).

To evaluate the functional effect of B cell-SQZ-mediated tolerance on T cell function, OT-I T cells restimulated with the SIINFEKL peptide (SEQ ID NO: 1) (CD8+ active OVA epitope) Percentage of splenic T cells expressing high levels of IFN-γ ( FIG. 3 , left), and IFN-γ production levels per cell ( FIG. 3 , right; MFI based on TCRVa2+/CD45.2+/CD44hi cells) was evaluated by intracellular cytokine staining for all groups. OT-I T cells from B cell-SQZ mice, as well as B cell-Endo treated mice (*** P <0.0005, **** P <0.0001), as well as IFN-γ high expressing cells compared to challenge controls showed a decrease in both the percentage of and IFN-γ production (*** P < 0.0005). Interestingly, B cell-SQZ mice even showed a significant decrease in the percentage of cells with high IFN-γ when compared to tolerant controls (* P < 0.05). The number of OT-I T cells producing IFN-γ was further confirmed by ELI spot (FIG. 4). Correlating with flow cytometry data, B cell-SQZ animals had significantly lower numbers of IFN-γ- when compared to challenge controls (** P < 0.01) and B cell-Endo (**** P < 0.0001). Positive T cells were produced.

Representative flow cytograms for TCRVa2+ splenic OT-I and lymph node OT-II T cells for each treatment group show that B cell-SQZ mice have higher numbers of OT-I and OT-II when compared to challenge controls and B cell-Endo. lower (Fig. 5, Table 2). Representative ICS flow cytograms for OT-I cells with high IFN-γ and CD44 antigen-activation markers show lower numbers of T cells with high IFN-γ in B cell-SQZ samples compared to challenge controls and B cell-Endo. notes (Fig. 6, Table 3).

Table 2

Table 3

Example 3: Constriction-mediated delivery of antigens to T cells to induce tolerance

To assess the ability of T cells antigen-presenting cells (APCs) containing intracellular antigens produced by constriction-mediated delivery of antigens to induce antigen-dependent tolerance in vivo, the number of antigen-specific T cells and Levels of the inflammatory cytokine IFN-γ were measured by flow cytometry.

materials and methods

On day 0, 2.5x10 ⁶ OVA-specific OT-I T cells per mouse were transferred adoptively from female OT-I donor mice (CD45.2) to C57BL/6J recipient mice (CD45.1). On the same day, 5x10 ⁶ T APC cells (CD90.1), T cells incubated in the presence of an immunogenic OVA epitope (SIINFEKL peptide, SEQ ID NO: 1) without stricture or with stricture-mediated transfer of the entire OVA protein, were also transfected. injected into mice. Compared to naïve (no T APC cells, no antigen), challenge animals were challenged with antigen on day 8 with intradermal injection of 10 μg OVA protein (antigen) + 50 ng LPS (adjuvant)/mouse. OT-I T cell responses to antigen challenge were measured on day 12 by flow cytometry for OT-I T cell numbers and IFN-γ intracellular staining. A schematic diagram of the treatment schedule is presented in FIG. 7 and the treatment groups are summarized in Table 4.

Table 4

result

The number of OT-I-specific T cells was determined on day 12 in mice from the four treatment groups. Activation and proliferation of OT-I T cells in both the draining lymph nodes and spleen were significantly inhibited in mice primed with T cells with OVA protein stenosis-mediated transfer when compared to challenge controls (**** P < 0.0001 ) (Fig. 8). These mice displayed similar OT-I T cell numbers to naïve mice or free OVA tolerant controls.

To evaluate the functional effect of T cell-SQZ APC-mediated tolerance on OT-I specific T cell function, IFN-γ production levels of OT-I T cells restimulated with free SIINFEKL peptide were evaluated for all groups (FIG. 9). OT-I T cells from T cell-SQZ mice showed a large decrease in the percentage of IFN-γ high expressing cells when compared to challenge controls, similar to tolerance controls (**** P <0.0001) ( **** P <0.0001).

Representative flow cytograms for OT-I T cell counts versus CD8+ T cells (FIG. 10, top panel) and percentage of cells with high IFN-γ versus CD44 antigen-activation marker (FIG. 10, bottom panel) compared to challenge controls. T cell-SQZ animals had lower OT-I T cell numbers and IFN-γ production (Table 5).

table 5

Example 4A: Prevention in Murine Type I Diabetes Model

Introduction

To determine the ability of antigen presenting cells (APCs - eg B and/or T cells) containing antigen delivered by SQZ to induce antigen-dependent tolerance in a prophylactic in vivo model of murine type I diabetes, tolerance Blood glucose levels are measured weekly over time after induction.

materials and methods

NOD/ShiltJ mice are treated with APC at 10 weeks of age. On day 0, APCs from NOD/ShiltJ mice were harvested and incubated with insulin B-chain peptide 9-23 (InsB9-23: CKKGSSHLVEALYLVCGERG, SEQ ID NO: 2) without constriction, or InsB9-23 with SQZ After delivery by condition, 5 M APC/mouse is injected into recipient mice. Control animals are injected with PBS (challenge control). Beginning on day 7 (11 weeks of age) after treatment, weekly blood glucose measurements are performed. A mouse is considered diabetic when blood glucose levels exceed 260 mg/dL as measured by a Bayer Contour Diabetes Meter/Glucose Test Strip. A schematic diagram of a representative treatment schedule is presented in FIG. 11A and the treatment groups are summarized in Table 6.

table 6

Example 4B: Treatment in Murine Type I Diabetes

Introduction

To determine the ability of antigen presenting cells (APCs - eg B and/or T cells) containing antigen delivered by SQZ to induce antigen-dependent tolerance in a therapeutic in vivo model of murine type I diabetes, mice Blood glucose levels are measured weekly over time after induction of tolerance at .

materials and methods

To induce rapid onset of T1D in recipients, CD4 T cells are harvested from BDC2.5 NOD mice, activated ex vivo with p31 mimetope peptide (YVRPLWVRME, SEQ ID NO: 3) for 4 days, and euglycemic NOD Adoptive transfer into recipients (5 M cells/mouse). Begin tolerance induction 8 hours after adoptive transfer and repeat every 3 days for a total of 3 doses. Recipients are treated with PBS (challenge control) or 5 M APC incubated with p31 (APC-Endo) or to which p31 was delivered by SQZ (APC-SQZ). Blood glucose is measured daily by Bayer Contour Diabetes Meter/Glucose Test Strips. Mice are considered diabetic when blood glucose levels exceed 260 mg/dL. A schematic diagram of a representative treatment schedule is presented in FIG. 11B and the treatment groups are summarized in Table 7.

table 7

Example 5A: Prevention in Murine MS-type Autoimmune Disorders

Introduction

To determine the ability of antigen presenting cells (APCs - eg B and/or T cells) containing antigen delivered by SQZ to induce antigen-dependent tolerance in an in vivo preventive model of murine MS-type autoimmune disorders , and assess daily clinical mobility scores over time.

materials and methods

Female C57BL/6 mice (10-12 weeks old) are treated with APC prior to induction of experimental autoimmune encephalomyelitis (EAE). On day -7, APCs from C57BL/6 mice were harvested and APCs were incubated with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG _35-55 : MEVGWYRSPFSRVVHLYRNGKGS, SEQ ID NO: 4) without constriction, or or MOG _35-55 or OVA _323-339 peptides (negative control: ISQAVHAAHAEINEAGRGS, SEQ ID NO: 5) are delivered to APCs by SQZ conditions, followed by injection of 5 M APC/mouse into recipient mice. On day 0, EAE is induced by administration of MOG _35-55 in CFA and pertussis toxin (Hooke kit) in PBS. Mice are scored daily starting on day 7, and clinical scores are defined as follows: 1, tail droop; 2, partial hind limb paralysis; 3, complete hind limb paralysis; 4, complete hindlimb paralysis and partial forelimb paralysis; and 5, moribund. A schematic diagram of a representative treatment schedule is presented in FIG. 12A and the treatment groups are summarized in Table 8.

Table 8

Example 5B: Treatment in Murine Autoimmune Disorders Type MS

Introduction

To determine the ability of antigen presenting cells (APCs - eg B and/or T cells) containing antigen delivered by SQZ to induce antigen-dependent tolerance in an established murine MS-type autoimmune disorder in vivo model For this purpose, clinical mobility scores are assessed daily over time.

materials and methods

In female C57BL/6 mice (10-12 weeks old), experimental autoimmune encephalomyelitis (EAE) is induced on day 0 by administration of MOG _35-55 in CFA and pertussis toxin (Hook kit) in PBS. Mice are then treated with APC on the day of onset of EAE, which occurs if the mouse scores ≧1 based on the following mobility criteria. On days -11/12, B cells from C57BL/6 mice were harvested and incubated with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG _35-55 : MEVGWYRSPFSRVVHLYRNGKGS, SEQ ID NO: 4) without constriction or , or MOG _35-55 or OVA _323-339 peptides (negative control: ISQAVHAAHAEINEAGRGS, SEQ ID NO: 5) are delivered to APCs by SQZ conditions, followed by injection of 5 M APC/mouse into recipient mice. Mice are scored daily starting on day 19, and clinical scores are defined as follows: 1, tail droop; 2, partial hind limb paralysis; 3, complete hind limb paralysis; 4, complete hindlimb paralysis and partial forelimb paralysis; and 5, moribund. A schematic diagram of a representative treatment schedule is presented in FIG. 12B and the treatment groups are summarized in Table 9.

Table 9

sequence listing

SEQUENCE LISTING <110> SQZ BIOTECHNOLOGIES COMPANY LOUGHEAD, Scott GILBERT, Jonathan B. BERNSTEIN, Howard SHAREI, Armon R. MOORE, Finola <120> INTRACELLULAR DELIVERY OF BIOMOLECULES TO INDUCE TOLERANCE <130> 75032-20012.40 <140> PCT/US2017/030933 <141> 2017-05-03 <150> US 62/331,384 <151> 2016-05-03 <160> 5 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 8 <212> PRT <213> artificial sequence <220> <223> synthetic construction <400> 1 Ser Ile Ile Asn Phe Glu Lys Leu 1 5 <210> 2 <211> 20 <212> PRT <213> artificial sequence <220> <223> synthetic construction <400> 2 Cys Lys Lys Gly Ser Ser His Leu Val Glu Ala Leu Tyr Leu Val 1 5 10 15 Cys Gly Glu Arg Gly 20 <210> 3 <211> 10 <212> PRT <213> artificial sequence <220> <223> synthetic construction <400> 3 Tyr Val Arg Pro Leu Trp Val Arg Met Glu 1 5 10 <210> 4 <211> 23 <212> PRT <213> artificial sequence <220> <223> synthetic construction <400> 4 Met Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val His 1 5 10 15 Leu Tyr Arg Asn Gly Lys Gly Ser 20 <210> 5 <211> 19 <212> PRT <213> artificial sequence <220> <223> synthetic construction <400> 5 Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala 1 5 10 15 Gly Arg Gly Ser 19

Claims (189)

An immune cell comprising an antigen for use in a method of suppressing an immune response or inducing tolerance in a subject in need thereof, wherein the method comprises:
a. A step of passing a cell suspension containing immune cells through a constriction, wherein the constriction causes disruption of the immune cells by modifying the immune cells, so that antigens and tolerogenic factors enter the immune cells through the disruption when they come into contact with the immune cells. and wherein the constriction has a width of 2 μm to 10 μm; and
b. Introducing immune cells into a subject
including,
wherein the presentation of the antigen by an immune cell suppresses an immune response to the antigen or induces tolerance to the antigen, the immune cell is an antigen-presenting cell, and the tolerogenic factor is arginase-1 (ARG1), oxidation nitrogen (NO), nitric oxide synthase 2 (NOS2), indolamine 2,3-dioxygenase (IDO), IL-4, IL-10, IL-13, TGF-β, IL-35, or IFNα immune cells. The immune cell of claim 1, wherein the antigen-presenting cell is a T cell, B cell, dendritic cell, monocyte, macrophage, NK cell, innate lymphoid cell, neutrophil, basophil, eosinophil, bone marrow derived suppressor cell, or mast cell. . 3. The immune cell according to claim 1 or 2, which is from the individual or a different individual. 3. The immune cell according to claim 1 or 2, wherein the constriction size is 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the diameter of the immune cell. 3. The method of claim 1 or 2, wherein the cell suspension is
(a) comprises a mixed cell population;
(b) is whole blood;
(c) comprising peripheral blood mononuclear cells.
immune cells. 3. The immune cell of claim 1 or 2, wherein the cell suspension comprises a purified cell population. 3. The immune cell of claim 1 or 2, wherein the cell suspension comprises human cells. The method of claim 1 or 2, wherein the antigen
(a) foreign antigen; or
(b) self-antigen
immune cells. The method of claim 1 or 2, wherein the antigen
(a) food allergens;
(b) antigens derived from transplanted tissue; or
(c) a therapeutic
immune cells. 10. The method of claim 9, wherein the therapeutic agent
(a) clotting factors;
(b) antibodies;
(c) growth factors;
(d) hormones; or
(e) recombinant cytokines
one or more of the immune cells. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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